CROSS-REFERENCE TO RELATED APPLICATIONS
REFERENCE TO SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted in ASCII
format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII
Copy, created on October 28, 2019, is named "GDS_0110PC_20191028_Seq_Listing_ST25"
and is 56,087 bytes in size.
BACKGROUND
[0003] Malaria is a serious disease caused by intra-erythrocyte parasites of the genus of
Plasmodium. The parasites are contracted via an infected female
Anopheles mosquito bite. There are five species of the parasite that cause malaria in humans:
P. falciparum, P. knowlesi, P. malariae, P. ovale and
P. vivax. According to the World Health Organization (WHO) there were 216 million cases of
Malaria worldwide in 2016, and of those, 445,000 were fatal.
[0004] The United States Food and Drug Administration (U.S. FDA) has implemented strict
blood screening guidelines for accepting or deferring donors who have travelled to
malaria-endemic regions. Travelers are deferred for one year after traveling to an
endemic region, or three years if the donor is a former resident of an endemic region.
People who have been diagnosed with malaria are deferred for three years after the
completion of treatment and symptom free. There are no approved tests available in
the U.S. to screen blood donations for
Plasmodium. This requires careful screening of prospective donors via a medical questionnaire.
[0005] In endemic countries, WHO recommends testing by thick blood films or using a highly
sensitive enzyme immunoassay. In non-endemic countries, WHO recommends donors are
deferred for six months from the last potential exposure combined with malaria antibody
testing using a highly sensitive enzyme immunoassay. Donors may be re-instated if
there is no evidence of malarial antibody. There is a risk that these methods are
not effective to detect low levels of parasitemia where transmission may occur.
CN102220419B and
WO2009074649 relate to detecting Plasmodium using amplification.
[0006] Transfused Transmitted Malaria (TTM) has been documented in the U.S. There have been
46 cases of TTM reported between 1911 and 2015 and the latest case was reported in
2018. There is a need for a specific and sensitive nucleic acid test (NAT) for detecting
Plasmodium species in a sample to reduce TTM and the number of deferred donors.
SUMMARY
[0007] The present invention is defined by the appended claims. In one aspect, the present
invention provides a method for specifically detecting
Plasmodium species nucleic acid in a sample. The method generally includes (1) contacting a
sample, the sample suspected of containing
Plasmodium species nucleic acid, with at least two oligomers for amplifying a target region
of a
Plasmodium species target nucleic acid, (2) performing an
in vitro nucleic acid amplification reaction, where any
Plasmodium target nucleic acid present in said sample is used as a template for generating an
amplification product, and (3) detecting the presence or absence of the amplification
product, thereby indicating the presence or absence of
Plasmodium species target nucleic acid in said sample. In some embodiments, the at least two
amplification oligomers comprise (a) an amplification oligomer comprising a target-hybridizing
sequence (i) that is from about 14 to about 20 contiguous nucleotides in length, is
contained in the sequence of SEQ ID NO: 162, and includes the sequence of SEQ ID NO:163,
or (ii) that is from about 14 to about 25 contiguous nucleotides in length, is contained
in the sequence of SEQ ID NO:166, and includes SEQ ID NO: 167 or SEQ ID NO: 168; and
(b) an amplification oligomer comprising a target-hybridizing sequence that is from
about 15 to about 33 contiguous nucleotides in length, is contained in SEQ ID NO:
169 and includes the sequence of SEQ ID NO:171, SEQ ID NO:172, or SEQ ID NO:173. Disclosed,
but not part of the claimed invention, is the use of at least two amplification oligomers
comprise (a') an amplification oligomer comprising a target-hybridizing sequence that
is contained in the sequence of SEQ ID NO:185 and includes the sequence of SEQ ID
NO:37, SEQ ID NO:46, or SEQ ID NO:187; and (b') an amplification oligomer comprising
a target-hybridizing sequence that is contained in the sequence of SEQ ID NO:188 and
includes the sequence of SEQ ID NO 83, SEQ ID NO:84, or SEQ ID NO:182.
[0008] In some embodiments of a method as above where the at least two amplification oligomers
comprise the amplification oligomers of (a)(i) and (b), the target-hybridizing sequence
of (a)(i) is selected from SEQ ID NOs:21, 23-25, 32, 33, 35, 54, and 55. In other
embodiments, the target-hybridizing sequence of (a)(i) is contained in the sequence
of SEQ ID NO: 164 and includes the sequence of SEQ ID NO:165; in some such variations,
the target-hybridizing sequence of (a)(i) is selected from SEQ ID NOs:21, 23-25, 32,
33, and 35.
[0009] In some embodiments of a method as above where the at least two amplification oligomers
comprise the amplification oligomers of (a)(ii) and (b), the target-hybridizing sequence
of (a)(ii) includes the sequence of SEQ ID NO:167; in some such variations, the target-hybridizing
sequence of (a)(ii) is selected from the SEQ ID NOs:28-31, 34, 40, 41, and 49-51.
In other embodiments, the target-hybridizing sequence of (a)(ii) includes the sequence
of SEQ ID NO:168; in some such variations, the target-hybridizing sequence of (a)(ii)
is selected from SEQ ID NOs:38, 39, 43, 44, and 53.
[0010] In some embodiments of a method as above where the at least two amplification oligomers
comprise the amplification oligomers of (a) and (b), the target-hybridizing sequence
of (b) is selected from SEQ ID NOs:80-82 and 85-100. In other embodiments, the target-hybridizing
sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence
of SEQ ID NO:171 or SEQ ID NO:172. In some variations where the target-hybridizing
sequence of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence
of SEQ ID NO:171, the target-hybridizing sequence of (b) is selected from SEQ ID NOs:81,
82, 85, 87-90, 94, and 96-98. In some variations where the target-hybridizing sequence
of (b) is contained in the sequence of SEQ ID NO: 170 and includes the sequence of
SEQ ID NO: 172, the target-hybridizing sequence of (b) is selected from SEQ ID NOs:80,
82, 85, and 87-100.
[0011] Disclosed, but not part of the claimed invention is the use of at least two amplification
oligomers comprise the amplification oligomers of (a') and (b'), the target-hybridizing
sequence of (a') is selected from SEQ ID NOs:37, 46, 183, and 184. In other embodiments,
the target-hybridizing sequence of (a') is contained in the sequence of SEQ ID NO:
186; in some such variations, the target-hybridizing sequence of (a') is SEQ ID NO:
183 or SEQ ID NO:184. In certain embodiments where the at least two amplification
oligomers comprise the amplification oligomers of (a') and (b'), the target-hybridizing
sequence of (b') is selected from SEQ ID NOs:83, 84, and 182. In more particular variations,
the target-hybridizing sequence of (a') is SEQ ID NO: 183 or SEQ ID NO: 184 and the
target-hybridizing sequence of (b') is SEQ ID NO: 182.
[0012] In some embodiments of a method as above, the amplification oligomer of (b) or (b')
is a promoter primer or promoter provider further comprising a promoter sequence located
5' to the target-hybridizing sequence of (b) or (b'), respectively. A particularly
suitable promoter sequence is a T7 promoter sequence such as,
e.g., SEQ ID NO:179. In specific variations where an amplification oligomer of (b) includes
a promoter sequence, the amplification oligomer of (b) comprises a sequence selected
from SEQ ID NOs:57-59 and 62-77. In specific variations where an amplification oligomer
of (b') includes a promoter sequence, the amplification oligomer of (b') comprises
a sequence selected from SEQ ID NOs:60, 61, and 181.
[0013] Particularly suitable pairs of amplification oligomer target-hybridizing sequences
of (a) and (b), respectively, are (A) SEQ ID NO:30 and SEQ ID NO:82, (B) SEQ ID NO:33
and SEQ ID NO:82, (C) SEQ ID NO:49 and SEQ ID NO:82, (D) SEQ ID NO:21 and SEQ ID NO:89,
(E) SEQ ID NO:30 and SEQ ID NO:89, (F) SEQ ID NO:33 and SEQ ID NO:89, (G) SEQ ID NO:49
and SEQ ID NO:89, (H) SEQ ID NO:21 and SEQ ID NO:92, (I) SEQ ID NO:30 and SEQ ID NO:92,
(J) SEQ ID NO 21 and SEQ ID NO:94, (K) SEQ ID NO:34 and SEQ ID NO:94, (L) SEQ ID NO:53
and SEQ ID NO:94, (M) SEQ ID NO:21 and SEQ ID NO:95, (N) SEQ ID NO:34 and SEQ ID NO:95,
and (O) SEQ ID NO:53 and SEQ ID NO:95. In some such embodiments, the amplification
oligomer of (b) is a promoter primer or promoter provider further comprising a promoter
sequence (for example, a T7 promoter sequence such as,
e.g., SEQ ID NO:179) located 5' to the target-hybridizing sequence of (b).
[0014] In some embodiments of a method as above where the at least two amplification oligomers
comprise the amplification oligomers of (a) and (b), the at least two amplification
oligomers comprise first and second amplification oligomers as in (a). In some such
variations, the at least two amplification oligomers comprise first and second amplification
oligomers as in (a)(ii). Particularly suitable first and second amplification oligomers
of (a)(ii) include a first amplification oligomer comprising a target-hybridizing
sequence that includes the sequence of SEQ ID NO:167
(e.g., the target-hybridizing sequence of SEQ ID NO:34) and a second amplification oligomer
comprising a target-hybridizing sequence that includes the sequence of SEQ ID NO:168
(e.g., the target-hybridizing sequence of SEQ ID NO:53).
[0015] In other embodiments of a method as above where the at least two amplification oligomers
comprise the amplification oligomers of (a) and (b), the at least two amplification
oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer
as in (a)(ii). In some such embodiments, the amplification oligomer as in (a)(i) comprises
a target-hybridizing sequence that is contained in the sequence of SEQ ID NO:164 and
includes the sequence of SEQ ID NO:165 (
e.g., the target-hybridizing sequence of SEQ ID NO:21), and the amplification oligomer
as in (a)(ii) comprises a target-hybridizing sequence that includes the sequence of
SEQ ID NO:167
(e.g., the target-hybridizing sequence of SEQ ID NO:34).
[0016] In some embodiments of a method as above where the at least two amplification oligomers
comprise the amplification oligomers of (a) and (b), the at least two amplification
oligomers comprise first and second amplification oligomers of (b). In some such embodiments,
each of the first and second amplification oligomers of (b) comprises a target-hybridizing
sequence that is contained in SEQ ID NO:170 and includes the sequence of SEQ ID NO:171
or SEQ ID NO:172
(e.g., a first amplification oligomer comprising the target-hybridizing sequence of SEQ
ID NO:94 and a second amplification oligomer comprising the target-hybridizing sequence
of SEQ ID NO:95). In some variations, each of the first and second amplification oligomers
of (b) is a promoter primer or promoter provider further comprising a promoter sequence
located 5' to the target-hybridizing sequence of (b). A particularly suitable promoter
sequence is a T7 promoter sequence such as,
e.g., SEQ ID NO:179. In specific variations where each of the first and second amplification
oligomers of (b) includes a promoter sequence, the first amplification oligomer of
(b) comprises the sequence of SEQ ID NO:71 and the second amplification oligomer of
(b) comprises the sequence of SEQ ID NO:72.
[0017] In certain embodiments of a method for detecting
Plasmodium species nucleic acid in a sample as above, the method further includes purifying
the target nucleic acid from other components in the sample before step (1). In some
such variations, the purifying step comprises contacting the sample with at least
one capture probe oligomer comprising a target-hybridizing sequence covalently attached
to a sequence or moiety that binds to an immobilized probe, where the target-hybridizing
sequence configured to specifically hybridize to the
Plasmodium species target nucleic acid. Particularly suitable capture probe oligomer target-hybridizing
sequences include sequences that are up to about 30 contiguous nucleotides in length
and include a sequence selected from SEQ ID NOs:11-15, 17, 19, and 20, including DNA
equivalents and DNA/RNA chimerics thereof. In more specific variations, the capture
probe oligomer target-hybridizing sequence is selected from SEQ ID NOs:11-15, 17,
19, and 20, including DNA equivalents and DNA/RNA chimerics thereof. In some embodiments,
the purifying step comprises contacting the sample with at least two capture probe
oligomers (
e.g., at least two capture probe oligomers as above); in some such variations, the at
least two capture probe oligomers include a first capture probe oligomer comprising
the target-hybridizing sequence of SEQ ID NO:19 and a second capture probe oligomer
comprising the target-hybridizing sequence of SEQ ID NO:20.
[0018] In some embodiments of a method for detecting
Plasmodium species nucleic acid in a sample as above, the detecting step (3) comprises contacting
the
in vitro nucleic acid amplification reaction with at least one detection probe oligomer comprising
a target-hybridizing sequence configured to specifically hybridize to the amplification
product under conditions whereby the presence or absence of the amplification product
is determined, thereby indicating the presence or absence of
Plasmodium species in the sample. In some such embodiments where the at least two amplification
oligomers comprise the amplification oligomers of (a) and (b), the detection probe
oligomer target-hybridizing sequence is from about 13 to about 40 nucleotides in length
and is (i) contained in the sequence of SEQ ID NO: 196 or its complement, or a DNA
equivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from
SEQ ID NO: 175, SEQ ID NO:176, SEQ ID NO: 177, and SEQ ID NO:178, including complements,
DNA equivalents, and DNA/RNA equivalents thereof. Suitable detection probe oligomer
target-hybridizing sequences include SEQ ID NOs: 131, 132, 135, 140, 145, 147-157,
and 159-161, including complements, DNA equivalents, and DNA/RNA chimerics thereof.
In certain embodiments, the detection probe oligomer target-hybridizing sequence is
(i) contained in the sequence of SEQ ID NO:197 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO:174 or SEQ
ID NO:175, including complements, DNA equivalents, and DNA/RNA chimerics thereof;
in some such variations comprising the sequence of SEQ ID NO:174 or its complement,
or a DNA equivalent or DNA/RNA chimeric thereof, the detection probe oligomer target-hybridizing
sequence is selected from SEQ ID NOs: 148-155 and 159, including complements, DNA
equivalents, and DNA/RNA chimerics thereof; in other such variations comprising SEQ
ID NO:175 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, the
detection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs:
147, 156, 157, 160, and 161, including complements, DNA equivalents, and DNA/RNA chimerics
thereof. In certain embodiments, the detection probe oligomer target-hybridizing sequence
is (i) contained in the sequence of SEQ ID NO:196 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from SEQ ID NO:177
and SEQ ID NO:178, including complements, DNA equivalents, and DNA/RNA chimerics thereof;
in some such variations, the detection probe oligomer target-hybridizing sequence
is selected from SEQ ID NOs: 131, 132, 135, 140, 147, 156, 157, 160, and 161, including
complements, DNA equivalents, and DNA/RNA chimerics thereof.
[0019] In other embodiments of a method as above where the detecting step (3) comprises
contacting the
in vitro nucleic acid amplification reaction with at least one detection probe oligomer and
where the at least two amplification oligomers comprise the amplification oligomers
of (a') and (b'), the detection probe oligomer target-hybridizing sequence is at least
about 13 nucleotides in length and is (i) contained in the sequence of SEQ ID NO:
189 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and (ii) includes
a sequence selected from SEQ ID NO:190 and SEQ ID NO:191, including complements, DNA
equivalents, and DNA/RNA equivalents thereof. In some such variations, the detection
probe oligomer target-hybridizing sequence is selected from the SEQ ID NOs:125-130
and 143, including complements, DNA equivalents, and DNA/RNA chimerics thereof.
[0020] In particular variations of a method for detecting
Plasmodium species nucleic acid in a sample as above, where the detecting step (3) comprises
contacting the
in vitro nucleic acid amplification reaction with at least one detection probe oligomer and
where the at least two amplification oligomers comprise the amplification oligomers
of (a) and (b), the amplification oligomer target-hybridizing sequence of (a), the
amplification oligomer target-hybridizing sequence of (b), and the detection probe
oligomer target-hybridizing sequence, respectively, are (A) SEQ ID NO:30, SEQ ID NO:82,
and SEQ ID NO:151 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO: 151 or its complement; (B) SEQ ID NO:30, SEQ ID NO:82, and SEQ ID NO:157 or
its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complement;
(C) SEQ ID NO:33, SEQ ID NO:82, and SEQ ID NO: 155 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO: 155 or its complement; (D) SEQ ID NO:49, SEQ ID
NO:82, and SEQ ID NO:150 or its complement, or a DNA equivalent or DNA/RNA chimeric
of SEQ ID NO:150 or its complement; (E) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO:
155 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 155 or
its complement; (F) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO:148 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:148 or its complement; (G) SEQ
ID NO:21, SEQ ID NO:89, and SEQ ID NO: 152 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO: 152 or its complement; (H) SEQ ID NO:30, SEQ ID
NO:89; and SEQ ID NO: 148 or its complement, or a DNA equivalent or DNA/RNA chimeric
of SEQ ID NO: 148 or its complement; (I) SEQ ID NO:30, SEQ ID NO:89; and SEQ ID NO:
152 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:152 or
its complement; (I) SEQ ID NO:33, SEQ ID NO:89; and SEQ ID NO:158 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:158 or its complement; (K) SEQ
ID NO:49, SEQ ID NO:89, and SEQ ID NO: 150 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO:150 or its complement; (L) SEQ ID NO:21, SEQ ID NO:92,
and SEQ ID NO:148 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO:148 or its complement; (M) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO:152 or
its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:152 or its complement;
(N) SEQ ID NO:30, SEQ ID NO:92; and SEQ ID NO: 148 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO: 148 or its complement; (O) SEQ ID NO:30, SEQ ID
NO:92; and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA/RNA chimeric
of SEQ ID NO:152 or its complement; (P) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO:
148 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:148 or
its complement; (Q) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO:152 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:152 or its complement; (R) SEQ
ID NO:34, SEQ ID NO:94, and SEQ ID NO:148 or its complement, or a DNA equivalent or
DNA/RNA chimeric of SEQ ID NO:148 or its complement; (S) SEQ ID NO:34, SEQ ID NO:94,
and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO: 152 or its complement; (T) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO:157 or
its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:157 or its complement;
(U) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 148 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO:148 or its complement; (V) SEQ ID NO:53, SEQ ID NO:94,
and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO:152 or its complement; (W) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO:157 or
its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:157 or its complement;
(X) SEQ ID NO:21, SEQ ID NO:95, and SEQ ID NO: 148 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO: 148 or its complement; (Y) SEQ ID NO:21, SEQ ID
NO:95, and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA/RNA chimeric
of SEQ ID NO:152 or its complement; (Z) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:
148 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:148 or
its complement; (AA) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO:152 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:152 or its complement; (AB) SEQ
ID NO:34, SEQ ID NO:95, and SEQ ID NO:157 or its complement, or a DNA equivalent or
DNA/RNA chimeric of SEQ ID NO: 157 or its complement; (AC) SEQ ID NO:53, SEQ ID NO:95,
and SEQ ID NO: 148 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO: 148 or its complement; (AD) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 152
or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:152 or its
complement; or (AE) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 157 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 157 or its complement.
[0021] In particular variations of a method for detecting
Plasmodium species nucleic acid in a sample as above, where the detecting step (3) comprises
contacting the
in vitro nucleic acid amplification reaction with at least one detection probe oligomer and
where the at least two amplification oligomers comprise the amplification oligomers
of (a') and (b'), the amplification oligomer target-hybridizing sequence of (a'),
the amplification oligomer target-hybridizing sequence of (b'), and the detection
probe oligomer target-hybridizing sequence, respectively, are (A) SEQ ID NO:183, SEQ
ID NO:182, and SEQ ID NO: 126 or its complement, or a DNA equivalent or DNA/RNA chimeric
of SEQ ID NO: 126 or its complement; (B) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID
NO: 127 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 127
or its complement; (C) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 128 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 128 or its complement; (D) SEQ
ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 143 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO: 143 or its complement; (E) SEQ ID NO: 183, SEQ ID
NO: 182, and SEQ ID NO: 129 or its complement, or a DNA equivalent or DNA/RNA chimeric
of SEQ ID NO: 129 or its complement; or (F) SEQ ID NO: 184, SEQ ID NO: 182, and SEQ
ID NO: 126 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:
126 or its complement.
[0022] In some embodiments of a method as above utilizing at least one detection probe oligomer,
the detection probe oligomer comprises a 2' methoxy modification on at least one of
a nucleotide residue member of the detection probe oligomer nucleotide sequence.
[0023] In some embodiments of a method as above utilizing at least one detection probe oligomer,
the detection probe oligomer further includes a detectable label such as, for example,
a fluorescent or chemiluminescent label. A particularly suitable chemiluminescent
label is a chemiluminescent acridinium ester (AE) compound linked between two nucleobases
of the detection probe oligomer. In some embodiments comprising a detectably labeled
probe oligomer, the detectable label is a fluorescent label and the detection probe
oligomer further includes a non-fluorescent quencher.
[0024] In some embodiments of a method as above, the detecting step (3) occurs during the
amplifying step (2). In some such embodiments, the method utilizes a detection probe
oligomer comprising a fluorescent label and a quencher (e.g., a molecular torch, a
molecular beacon, or a TaqMan detection probe).
[0025] In some embodiments of a method as above utilizing at least one detection probe oligomer,
the detection probe further includes a non-target-hybridizing sequence. In particular
variations, a detection probe oligomer comprising a non-target-hybridizing sequence
is a hairpin detection probe such as,
e.g., a molecular beacon or a molecular torch.
[0026] In certain embodiments, a method for detecting
Plasmodium species nucleic acid in a sample as above utilizes at least two detection probe oligomers.
In some such embodiments, the at least two detection probe oligomers comprise first
and second detection probe oligomers, where (A) the first detection probe oligomer
comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ
ID NO: 197 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and
(ii) includes the sequence of SEQ ID NO: 175 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, and (B) the second detection probe oligomer comprises
a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO:
197 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and (ii) includes
the sequence of SEQ ID NO: 176 or its complement, or a DNA equivalent or DNA/RNA chimeric
thereof. In a more specific variations, the first detection probe oligomer comprises
the target-hybridizing sequence of SEQ ID NO: 157 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, and the second detection probe oligomer comprises a target-hybridizing
sequence selected from SEQ ID NO: 148 and SEQ ID NO:152, including complements, DNA
equivalents, and DNA/RNA chimerics thereof.
[0027] In certain variations of a method for detecting
Plasmodium species nucleic acid in a sample as above, the
in vitro nucleic acid amplification reaction at step (2) is an isothermal amplification reaction
(e.g., a transcription-mediated amplification (TMA) reaction).
[0028] In certain variations of a method for detecting
Plasmodium species nucleic acid in a sample as above, the amplification reaction is a real-time
amplification reaction.
[0029] In some embodiments of a method for detecting
Plasmodium species nucleic acid in a sample as above, the sample is a clinical sample. In some
embodiments, the sample is a blood sample such as, for example, a red blood cell sample
(
e.g., a lysed blood cell sample or lysed red blood cell sample).
[0030] In another aspect, the present invention provides a combination of at least two oligomers
for determining the presence or absence of
Plasmodium species in a sample. The oligomer combination generally includes at least two oligomers
for amplifying a target region
of Plasmodium species target nucleic acid. In some embodiments, the at least two amplification
oligomers comprise (a) an amplification oligomer comprising a target-hybridizing sequence
(i) that is from about 14 to about 20 contiguous nucleotides in length, is contained
in the sequence of SEQ ID NO: 162, and includes the sequence of SEQ ID NO: 163, or
(ii) that is from about 14 to about 25 contiguous nucleotides in length, is contained
in the sequence of SEQ ID NO: 166, and includes SEQ ID NO: 167 or SEQ ID NO: 168;
and (b) an amplification oligomer comprising a target-hybridizing sequence that is
from about 15 to about 33 contiguous nucleotides in length, is contained in SEQ ID
NO: 169 and includes the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO:
173. Disclosed, but not part of the claimed invention is the use of at least two amplification
oligomers comprise (a') an amplification oligomer comprising a target-hybridizing
sequence that is contained in the sequence of SEQ ID NO: 185 and includes the sequence
of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO: 187; and (b') an amplification oligomer
comprising a target-hybridizing sequence that is contained in the sequence of SEQ
ID NO: 188 and includes the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:
182.
[0031] In some embodiments of an oligomer combination as above where the at least two amplification
oligomers comprise the amplification oligomers of (a)(i) and (b), the target-hybridizing
sequence of (a)(i) is selected from SEQ ID NOs:21, 23-25, 32, 33, 35, 54, and 55.
In other embodiments, the target-hybridizing sequence of (a)(i) is contained in the
sequence of SEQ ID NO: 164 and includes the sequence of SEQ ID NO: 165; in some such
variations, the target-hybridizing sequence of (a)(i) is selected from SEQ ID NOs:21,
23-25, 32, 33, and 35.
[0032] In some embodiments of an oligomer combination as above where the at least two amplification
oligomers comprise the amplification oligomers of (a)(ii) and (b), the target-hybridizing
sequence of (a)(ii) includes the sequence of SEQ ID NO: 167; in some such variations,
the target-hybridizing sequence of (a)(ii) is selected from the SEQ ID NOs:28-31,
34, 40, 41, and 49-51. In other embodiments, the target-hybridizing sequence of (a)(ii)
includes the sequence of SEQ ID NO: 168; in some such variations, the target-hybridizing
sequence of (a)(ii) is selected from SEQ ID NOs:38, 39, 43, 44, and 53.
[0033] In some embodiments of an oligomer combination as above where the at least two amplification
oligomers comprise the amplification oligomers of (a) and (b), the target-hybridizing
sequence of (b) is selected from SEQ ID NOs:80-82 and 85-100 In other embodiments,
the target-hybridizing sequence of (b) is contained in the sequence of SEQ ID NO:
170 and includes the sequence of SEQ ID NO:171 or SEQ ID NO:172. In some variations
where the target-hybridizing sequence of (b) is contained in the sequence of SEQ ID
NO: 170 and includes the sequence of SEQ ID NO: 171, the target-hybridizing sequence
of (b) is selected from SEQ ID NOs:81, 82, 85, 87-90, 94, and 96-98. In some variations
where the target-hybridizing sequence of (b) is contained in the sequence of SEQ ID
NO: 170 and includes the sequence of SEQ ID NO:172, the target-hybridizing sequence
of (b) is selected from SEQ ID NOs:80, 82, 85, and 87-100.
[0034] In some embodiments of an oligomer combination as above where the at least two amplification
oligomers comprise the amplification oligomers of (a') and (b'), the target-hybridizing
sequence of (a') is selected from SEQ ID NOs:37, 46, 183, and 184. In other embodiments,
the target-hybridizing sequence of (a') is contained in the sequence of SEQ ID NO:
186; in some such variations, the target-hybridizing sequence of (a') is SEQ ID NO:183
or SEQ ID NO:184. In certain embodiments where the at least two amplification oligomers
comprise the amplification oligomers of (a') and (b'), the target-hybridizing sequence
of (b') is selected from SEQ ID NOs:83, 84, and 182. In more particular variations,
the target-hybridizing sequence of (a') is SEQ ID NO:183 or SEQ ID NO:184 and the
target-hybridizing sequence of (b') is SEQ ID NO:182.
[0035] In some embodiments of an oligomer combination as above, the amplification oligomer
of (b) or (b') is a promoter primer or promoter provider further comprising a promoter
sequence located 5' to the target-hybridizing sequence of (b) or (b'), respectively.
A particularly suitable promoter sequence is a T7 promoter sequence such as,
e.g., SEQ ID NO: 179. In specific variations where an amplification oligomer of (b) includes
a promoter sequence, the amplification oligomer of (b) comprises a sequence selected
from SEQ ID NOs:57-59 and 62-77. In specific variations where an amplification oligomer
of (b') includes a promoter sequence, the amplification oligomer of (b') comprises
a sequence selected from SEQ ID NOs:60, 61, and 181.
[0036] Particularly suitable pairs of amplification oligomer target-hybridizing sequences
of (a) and (b), respectively, are (A) SEQ ID NO:30 and SEQ ID NO:82, (B) SEQ ID NO:33
and SEQ ID NO:82, (C) SEQ ID NO:49 and SEQ ID NO:82, (D) SEQ ID NO:21 and SEQ ID NO:89,
(E) SEQ ID NO:30 and SEQ ID NO:89, (F) SEQ ID NO:33 and SEQ ID NO:89, (G) SEQ ID NO:49
and SEQ ID NO:89, (H) SEQ ID NO:21 and SEQ ID NO:92, (I) SEQ ID NO:30 and SEQ ID NO:92,
(J) SEQ ID NO 21 and SEQ ID NO:94, (K) SEQ ID NO:34 and SEQ ID NO:94, (L) SEQ ID NO:53
and SEQ ID NO:94, (M) SEQ ID NO:21 and SEQ ID NO:95, (N) SEQ ID NO:34 and SEQ ID NO:95,
and (O) SEQ ID NO:53 and SEQ ID NO:95. In some such embodiments, the amplification
oligomer of (b) is a promoter primer or promoter provider further comprising a promoter
sequence (for example, a T7 promoter sequence such as,
e.g., SEQ ID NO: 179) located 5' to the target-hybridizing sequence of (b).
[0037] In some embodiments of an oligomer combination as above where the at least two amplification
oligomers comprise the amplification oligomers of (a) and (b), the at least two amplification
oligomers comprise first and second amplification oligomers as in (a). In some such
variations, the at least two amplification oligomers comprise first and second amplification
oligomers as in (a)(ii). Particularly suitable first and second amplification oligomers
of (a)(ii) include a first amplification oligomer comprising a target-hybridizing
sequence that includes the sequence of SEQ ID NO: 167
(e.g., the target-hybridizing sequence of SEQ ID NO:34) and a second amplification oligomer
comprising a target-hybridizing sequence that includes the sequence of SEQ ID NO:
168
(e.g., the target-hybridizing sequence of SEQ ID NO:53).
[0038] In other embodiments of an oligomer combination as above where the at least two amplification
oligomers comprise the amplification oligomers of (a) and (b), the at least two amplification
oligomers comprise an amplification oligomer as in (a)(i) and an amplification oligomer
as in (a)(ii). In some such embodiments, the amplification oligomer as in (a)(i) comprises
a target-hybridizing sequence that is contained in the sequence of SEQ ID NO: 164
and includes the sequence of SEQ ID NO: 165
(e.g., the target-hybridizing sequence of SEQ ID NO:21), and the amplification oligomer
as in (a)(ii) comprises a target-hybridizing sequence that includes the sequence of
SEQ ID NO: 167
(e.g., the target-hybridizing sequence of SEQ ID NO:34).
[0039] In some embodiments of an oligomer combination as above where the at least two amplification
oligomers comprise the amplification oligomers of (a) and (b), the at least two amplification
oligomers comprise first and second amplification oligomers of (b). In some such embodiments,
each of the first and second amplification oligomers of (b) comprises a target-hybridizing
sequence that is contained in SEQ ID NO: 170 and includes the sequence of SEQ ID NO:171
or SEQ ID NO:172
(e.g., a first amplification oligomer comprising the target-hybridizing sequence of SEQ
ID NO:94 and a second amplification oligomer comprising the target-hybridizing sequence
of SEQ ID NO:95). In some variations, each of the first and second amplification oligomers
of (b) is a promoter primer or promoter provider further comprising a promoter sequence
located 5' to the target-hybridizing sequence of (b). A particularly suitable promoter
sequence is a T7 promoter sequence such as,
e.g., SEQ ID NO:179. In specific variations where each of the first and second amplification
oligomers of (b) includes a promoter sequence, the first amplification oligomer of
(b) comprises the sequence of SEQ ID NO:71 and the second amplification oligomer of
(b) comprises the sequence of SEQ ID NO:72.
[0040] In certain embodiments of an oligomer combination for detecting
Plasmodium species nucleic acid in a sample as above, the oligomer combination further includes
at least one capture probe oligomer comprising a target-hybridizing sequence covalently
attached to a sequence or moiety that binds to an immobilized probe, where the target-hybridizing
sequence configured to specifically hybridize to the
Plasmodium species target nucleic acid. Particularly suitable capture probe oligomer target-hybridizing
sequences include sequences that are up to about 30 contiguous nucleotides in length
and include a sequence selected from SEQ ID NOs: 11-15, 17, 19, and 20, including
DNA equivalents and DNA/RNA chimerics thereof. In more specific variations, the capture
probe oligomer target-hybridizing sequence is selected from SEQ ID NOs:11-15, 17,
19, and 20, including DNA equivalents and DNA/RNA chimerics thereof. In some embodiments,
the oligomer combination includes at least two capture probe oligomers
(e.g., at least two capture probe oligomers as above); in some such variations, the at least
two capture probe oligomers include a first capture probe oligomer comprising the
target-hybridizing sequence of SEQ ID NO:19 and a second capture probe oligomer comprising
the target-hybridizing sequence of SEQ ID NO:20.
[0041] In some embodiments of an oligomer combination for detecting
Plasmodium species nucleic acid in a sample as above, the oligomer combination further includes
at least one detection probe oligomer comprising a target-hybridizing sequence configured
to specifically hybridize to a
Plasmodium species amplicon amplifiable by the at least two amplification oligomers. In some
such embodiments where the at least two amplification oligomers comprise the amplification
oligomers of (a) and (b), the detection probe oligomer target-hybridizing sequence
is from about 13 to about 40 nucleotides in length and is (i) contained in the sequence
of SEQ ID NO:196 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof,
and (ii) includes a sequence selected from SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177,
and SEQ ID NO:178, including complements, DNA equivalents, and DNA/RNA equivalents
thereof. Suitable detection probe oligomer target-hybridizing sequences include SEQ
ID NOs:131, 132, 135, 140, 145, 147-157, and 159-161, including complements, DNA equivalents,
and DNA/RNA chimerics thereof. In certain embodiments, the detection probe oligomer
target-hybridizing sequence is (i) contained in the sequence of SEQ ID NO:197 or its
complement, or a DNA equivalent or DNA/RNA chimeric thereof, and (ii) includes the
sequence of SEQ ID NO:174 or SEQ ID NO:175, including complements, DNA equivalents,
and DNA/RNA chimerics thereof; in some such variations comprising the sequence of
SEQ ID NO: 174 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof,
the detection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs:
148-155 and 159, including complements, DNA equivalents, and DNA/RNA chimerics thereof;
in other such variations comprising SEQ ID NO:175 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, the detection probe oligomer target-hybridizing sequence
is selected from SEQ ID NOs:147, 156, 157, 160, and 161, including complements, DNA
equivalents, and DNA/RNA chimerics thereof. In certain embodiments, the detection
probe oligomer target-hybridizing sequence is (i) contained in the sequence of SEQ
ID NO:196 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and
(ii) includes a sequence selected from SEQ ID NO:177 and SEQ ID NO:178, including
complements, DNA equivalents, and DNA/RNA chimerics thereof; in some such variations,
the detection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs:131,
132, 135, 140, 147, 156, 157, 160, and 161, including complements, DNA equivalents,
and DNA/RNA chimerics thereof.
[0042] In other embodiments of an oligomer combination as above further including at least
one detection probe oligomer and where the at least two amplification oligomers comprise
the amplification oligomers of (a') and (b'), the detection probe oligomer target-hybridizing
sequence is at least about 13 nucleotides in length and is (i) contained in the sequence
of SEQ ID NO:189 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof,
and (ii) includes a sequence selected from SEQ ID NO:190 and SEQ ID NO:191, including
complements, DNA equivalents, and DNA/RNA equivalents thereof. In some such variations,
the detection probe oligomer target-hybridizing sequence is selected from the SEQ
ID NOs: 125-130 and 143, including complements, DNA equivalents, and DNA/RNA chimerics
thereof.
[0043] In particular variations of an oligomer combination as above further including at
least one detection probe oligomer and where the at least two amplification oligomers
comprise the amplification oligomers of (a) and (b), the amplification oligomer target-hybridizing
sequence of (a), the amplification oligomer target-hybridizing sequence of (b), and
the detection probe oligomer target-hybridizing sequence, respectively, are (A) SEQ
ID NO:30, SEQ ID NO:82, and SEQ ID NO:151 or its complement, or a DNA equivalent or
DNA/RNA chimeric of SEQ ID NO: 151 or its complement; (B) SEQ ID NO:30, SEQ ID NO:82,
and SEQ ID NO: 157 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO: 157 or its complement; (C) SEQ ID NO:33, SEQ ID NO:82, and SEQ ID NO:155 or
its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:155 or its complement;
(D) SEQ ID NO:49, SEQ ID NO:82, and SEQ ID NO: 150 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO: 150 or its complement; (E) SEQ ID NO:49, SEQ ID
NO:82, and SEQ ID NO:155 or its complement, or a DNA equivalent or DNA/RNA chimeric
of SEQ ID NO:155 or its complement; (F) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO:
148 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 148 or
its complement; (G) SEQ ID NO:21, SEQ ID NO:89, and SEQ ID NO: 152 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complement; (H) SEQ
ID NO:30, SEQ ID NO:89; and SEQ ID NO:148 or its complement, or a DNA equivalent or
DNA/RNA chimeric of SEQ ID NO:148 or its complement; (I) SEQ ID NO:30, SEQ ID NO:89;
and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO:152 or its complement; (I) SEQ ID NO:33, SEQ ID NO:89; and SEQ ID NO:158 or
its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:158 or its complement;
(K) SEQ ID NO:49, SEQ ID NO:89, and SEQ ID NO: 150 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO:150 or its complement; (L) SEQ ID NO:21, SEQ ID NO:92,
and SEQ ID NO: 148 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO: 148 or its complement; (M) SEQ ID NO:21, SEQ ID NO:92, and SEQ ID NO: 152 or
its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:152 or its complement;
(N) SEQ ID NO:30, SEQ ID NO:92; and SEQ ID NO: 148 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO: 148 or its complement; (O) SEQ ID NO:30, SEQ ID
NO:92; and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA/RNA chimeric
of SEQ ID NO:152 or its complement; (P) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO:
148 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 148 or
its complement; (Q) SEQ ID NO:21, SEQ ID NO:94, and SEQ ID NO: 152 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complement; (R) SEQ
ID NO:34, SEQ ID NO:94, and SEQ ID NO:148 or its complement, or a DNA equivalent or
DNA/RNA chimeric of SEQ ID NO:148 or its complement; (S) SEQ ID NO:34, SEQ ID NO:94,
and SEQ ID NO:152 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO: 152 or its complement; (T) SEQ ID NO:34, SEQ ID NO:94, and SEQ ID NO:157 or
its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:157 or its complement;
(U) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO: 148 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO: 148 or its complement; (V) SEQ ID NO:53, SEQ ID
NO:94, and SEQ ID NO: 152 or its complement, or a DNA equivalent or DNA/RNA chimeric
of SEQ ID NO: 152 or its complement; (W) SEQ ID NO:53, SEQ ID NO:94, and SEQ ID NO:
157 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:157 or
its complement; (X) SEQ ID NO:21, SEQ ID NO:95, and SEQ ID NO: 148 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 148 or its complement; (Y) SEQ
ID NO:21, SEQ ID NO:95, and SEQ ID NO:152 or its complement, or a DNA equivalent or
DNA/RNA chimeric of SEQ ID NO:152 or its complement; (Z) SEQ ID NO:34, SEQ ID NO:95,
and SEQ ID NO: 148 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO:148 or its complement; (AA) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 152 or
its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 152 or its complement;
(AB) SEQ ID NO:34, SEQ ID NO:95, and SEQ ID NO: 157 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO:157 or its complement; (AC) SEQ ID NO:53, SEQ ID
NO:95, and SEQ ID NO:148 or its complement, or a DNA equivalent or DNA/RNA chimeric
of SEQ ID NO:148 or its complement; (AD) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO:152
or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:152 or its
complement; or (AE) SEQ ID NO:53, SEQ ID NO:95, and SEQ ID NO: 157 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO:157 or its complement.
[0044] In particular variations of an oligomer combination as above further including at
least one detection probe oligomer and where the at least two amplification oligomers
comprise the amplification oligomers of (a') and (b'), the amplification oligomer
target-hybridizing sequence of (a'), the amplification oligomer target-hybridizing
sequence of (b'), and the detection probe oligomer target-hybridizing sequence, respectively,
are (A) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO:126 or its complement, or a DNA
equivalent or DNA/RNA chimeric of SEQ ID NO: 126 or its complement; (B) SEQ ID NO:183,
SEQ ID NO: 182, and SEQ ID NO:127 or its complement, or a DNA equivalent or DNA/RNA
chimeric of SEQ ID NO: 127 or its complement; (C) SEQ ID NO: 183, SEQ ID NO: 182,
and SEQ ID NO: 128 or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ
ID NO: 128 or its complement; (D) SEQ ID NO:183, SEQ ID NO:182, and SEQ ID NO: 143
or its complement, or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 143 or its
complement; (E) SEQ ID NO: 183, SEQ ID NO: 182, and SEQ ID NO: 129 or its complement,
or a DNA equivalent or DNA/RNA chimeric of SEQ ID NO: 129 or its complement; or (F)
SEQ ID NO:184, SEQ ID NO: 182, and SEQ ID NO: 126 or its complement, or a DNA equivalent
or DNA/RNA chimeric of SEQ ID NO: 126 or its complement.
[0045] In some embodiments of an oligomer combination as above further including at least
one detection probe oligomer, the detection probe oligomer comprises a 2' methoxy
modification on at least one of a nucleotide residue member of the detection probe
oligomer nucleotide sequence.
[0046] In some embodiments of an oligomer combination as above further including at least
one detection probe oligomer, the detection probe oligomer further includes a detectable
label such as, for example, a fluorescent or chemiluminescent label. A particularly
suitable chemiluminescent label is a chemiluminescent acridinium ester (AE) compound
linked between two nucleobases of the detection probe oligomer. In some embodiments
comprising a detectably labeled probe oligomer, the detectable label is a fluorescent
label and the detection probe oligomer further includes a non-fluorescent quencher;
particularly suitable detection probe oligomers comprising a fluorescent label and
a quencher including molecular torches, molecular beacons, and TaqMan detection probes.
[0047] In some embodiments of an oligomer combination as above further including at least
one detection probe oligomer, the detection probe further includes a non-target-hybridizing
sequence. In particular variations, a detection probe oligomer comprising a non-target-hybridizing
sequence is a hairpin detection probe such as,
e.g., a molecular beacon or a molecular torch.
[0048] In certain embodiments, an oligomer combination for detecting
Plasmodium species nucleic acid in a sample as above includes at least two detection probe oligomers.
In some such embodiments, the at least two detection probe oligomers comprise first
and second detection probe oligomers, where (A) the first detection probe oligomer
comprises a target-hybridizing sequence that is (i) contained in the sequence of SEQ
ID NO: 197 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and
(ii) includes the sequence of SEQ ID NO:175 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, and (B) the second detection probe oligomer comprises
a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO:
197 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, and (ii) includes
the sequence of SEQ ID NO:176 or its complement, or a DNA equivalent or DNA/RNA chimeric
thereof. In a more specific variation, the first detection probe oligomer comprises
the target-hybridizing sequence of SEQ ID NO:157 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, and the second detection probe oligomer comprises a target-hybridizing
sequence selected from SEQ ID NO:148 and SEQ ID NO:152, including complements, DNA
equivalents, and DNA/RNA chimerics thereof.
[0049] In another aspect, the present invention provides use of a combination of at least
two oligomers as above for specifically amplifying
Plasmodium species nucleic acid in a sample.
[0050] In another aspect, the present invention provides a detection probe oligomer for
specifically detecting a
Plasmodium species target nucleic acid in a sample. In some embodiments, the detection probe
oligomer comprises a target-hybridizing sequence that is from about 13 to about 40
nucleotides in length and configured to specifically hybridize to a target sequence
contained within a
Plasmodium species target region amplifiable by an oligomer combination comprising first and
second
Plasmodium-specific amplification oligomers, where (a) the first amplification oligomer comprises a target-hybridizing
sequence (i) that is from about 14 to about 20 contiguous nucleotides in length, is
contained in the sequence of SEQ ID NO:162, and includes the sequence of SEQ ID NO:163,
or (ii) that is from about 14 to about 25 contiguous nucleotides in length, is contained
in the sequence of SEQ ID NO:166, and includes the sequence of SEQ ID NO:167 or SEQ
ID NO:168; and (b) the second amplification oligomer comprises a target-hybridizing
sequence that is from about 15 to about 33 contiguous nucleotides in length, is contained
in SEQ ID NO:169 and includes the sequence of SEQ ID NO:171, SEQ ID NO:172, or SEQ
ID NO:173. In some such embodiments, the detection probe oligomer target-hybridizing
sequence is (i) contained in the sequence of SEQ ID NO:196 or its complement, or a
DNA equivalent or DNA/RNA chimeric thereof, and (ii) includes a sequence selected
from SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, and SEQ ID NO:178, including complements,
DNA equivalents, and DNA/RNA equivalents thereof. Suitable detection probe oligomer
target-hybridizing sequences include SEQ ID NOs:131, 132, 135, 140, 145, 147-157,
and 159-161, including complements, DNA equivalents, and DNA/RNA chimerics thereof.
In certain embodiments, the detection probe oligomer target-hybridizing sequence is
(i) contained in the sequence of SEQ ID NO:197 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO:174 or SEQ
ID NO:175, including complements, DNA equivalents, and DNA/RNA chimerics thereof;
in some such variations comprising the sequence of SEQ ID NO:174 or its complement,
or a DNA equivalent or DNA/RNA chimeric thereof, the detection probe oligomer target-hybridizing
sequence is selected from SEQ ID NOs: 148-155 and 159, including complements, DNA
equivalents, and DNA/RNA chimerics thereof; in other such variations comprising SEQ
ID NO:175 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof, the
detection probe oligomer target-hybridizing sequence is selected from SEQ ID NOs:
147, 156, 157, 160, and 161, including complements, DNA equivalents, and DNA/RNA chimerics
thereof. In certain embodiments, the detection probe oligomer target-hybridizing sequence
is (i) contained in the sequence of SEQ ID NO:196 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from SEQ ID NO:177
and SEQ ID NO:178, including complements, DNA equivalents, and DNA/RNA chimerics thereof;
in some such variations, the detection probe oligomer target-hybridizing sequence
is selected from SEQ ID NOs: 131, 132, 135, 140, 147, 156, 157, 160, and 161, including
complements, DNA equivalents, and DNA/RNA chimerics thereof.
[0051] In other embodiments of a detection probe oligomer for specifically detecting a
Plasmodium species target nucleic acid in a sample, the detection probe oligomer comprises a
target-hybridizing sequence that is at least about 13 nucleotides in length and configured
to specifically hybridize to a target sequence contained within a
Plasmodium species target region amplifiable by an oligomer combination comprising first and
second
Plasmodium-specific amplification oligomers, where (a) the first amplification oligomer comprises a target-hybridizing
sequence that is contained in the sequence of SEQ ID NO:185 and includes the sequence
of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO:187; and (b) the second amplification
oligomer comprises a target-hybridizing sequence that is contained in SEQ ID NO:188
and includes the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:182. In some
such embodiments, the detection probe oligomer target-hybridizing sequence is (i)
contained in the sequence of SEQ ID NO:189 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, and (ii) includes a sequence selected from SEQ ID NO:
190 and SEQ ID NO:191, including complements, DNA equivalents, and DNA/RNA equivalents
thereof. In more specific variations, the detection probe oligomer target-hybridizing
sequence is selected from the SEQ ID NOs: 125-130 and 143, including complements,
DNA equivalents, and DNA/RNA chimerics thereof.
[0052] In some embodiments of a detection probe oligomer as above, the detection probe oligomer
comprises a 2' methoxy modification on at least one of a nucleotide residue member
of the detection probe oligomer nucleotide sequence.
[0053] In some embodiments of a detection probe oligomer as above, the detection probe oligomer
further includes a detectable label such as, for example, a fluorescent or chemiluminescent
label. A particularly suitable chemiluminescent label is a chemiluminescent acridinium
ester (AE) compound linked between two nucleobases of the detection probe oligomer.
In some embodiments comprising a detectable label, the detectable label is a fluorescent
label and the detection probe oligomer further includes a non-fluorescent quencher;
particularly suitable detection probe oligomers comprising a fluorescent label and
a quencher including molecular torches, molecular beacons, and TaqMan detection probes.
[0054] In some embodiments of a detection probe oligomer as above, the detection probe further
includes a non-target-hybridizing sequence. In particular variations, a detection
probe oligomer comprising a non-target-hybridizing sequence is a hairpin detection
probe such as,
e.g., a molecular beacon or a molecular torch.
[0055] In another aspect, the present invention provides a combination of at least two oligomers
for detecting a
Plasmodium species target nucleic acid in a sample, the oligomer combination comprising at least
two detection probe oligomers as above. In some embodiments, the at least two detection
probe oligomers comprise a (A) a first detection probe oligomer comprising a target-hybridizing
sequence that (i) is contained in the sequence of SEQ ID NO: 197 or its complement,
or a DNA equivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of
SEQ ID NO: 175 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof,
and (B) a second detection probe oligomer comprising a target-hybridizing sequence
that (i) is contained in the sequence of SEQ ID NO: 197 or its complement, or a DNA
equivalent or DNA/RNA chimeric thereof, and (ii) includes the sequence of SEQ ID NO:
176 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof. In more specific
variations, the first detection probe oligomer comprises the target-hybridizing sequence
of SEQ ID NO: 157 or its complement, or a DNA equivalent or DNA/RNA chimeric thereof,
and the second detection probe oligomer comprises a target-hybridizing sequence selected
from SEQ ID NO:148 and SEQ ID NO:152, including complements, DNA equivalents, and
DNA/RNA chimerics thereof.
[0056] In another aspect, the present invention provides use of a detection probe oligomer
or oligomer combination according as above for specifically detecting
Plasmodium species nucleic acid in a sample.
[0057] In another aspect, the present invention provides a capture probe oligomer for specifically
isolating
Plasmodium species nucleic acid from a sample. In some embodiments, the capture probe oligomer
comprises a target-hybridizing sequence covalently attached to a sequence or moiety
that binds to an immobilized probe, where the target-hybridizing sequence is up to
about 30 contiguous nucleotides in length and includes a sequence selected from SEQ
ID NOs: 11-15, 17, 19, and 20, including DNA equivalents and DNA/RNA chimerics thereof.
In more specification variations, the capture probe oligomer target-hybridizing sequence
is selected from SEQ ID NOs:11-15, 17, 19, and 20, including DNA equivalents and DNA/RNA
chimerics thereof.
[0058] In another aspect, the present invention provides a combination of at least two oligomers
for specifically isolating
Plasmodium species nucleic acid from a sample, the oligomer combination comprising at least
two capture probe oligomers as above. In some embodiments, the at least two capture
probe oligomers comprise a first capture probe oligomer comprising the target-hybridizing
sequence of SEQ ID NO:19, or a DNA equivalent or DNA/RNA chimeric thereof, and a second
capture probe oligomer comprising the target-hybridizing sequence of SEQ ID NO:20,
or a DNA equivalent or DNA/RNA chimeric thereof.
[0059] In another aspect, the present invention provides use of a capture probe oligomer
or oligomer combination as above for specifically capturing
Plasmodium species nucleic acid from a sample.
[0060] In another aspect, the present invention provides a kit comprising a combination
of at least two oligomers as above.
[0061] In another aspect, the present invention a reaction mixture comprising a combination
of at least two oligomers as above.
[0062] These and other aspects of the invention will become evident upon reference to the
following detailed description of the invention and the attached drawings.
DEFINITIONS
[0063] Unless defined otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art pertinent
to the methods and compositions described. As used herein, the following terms and
phrases have the meanings ascribed to them unless specified otherwise.
[0064] The terms "a," "an," and "the" include plural referents, unless the context clearly
indicates otherwise.
[0065] "Sample" includes any specimen that may contain, or is suspected of containing,
Plasmodium species or components thereof, such as nucleic acids or fragments of
Plasmodium nucleic acids. The sample may be an isolated sample. Samples include "biological
samples" which include any tissue or material derived from a living or dead human
that may contain the
Plasmodium parasite or components thereof
(e.g., a target nucleic acid derived therefrom), including,
e.g., blood, peripheral blood and red blood cells. The use of other sample types that
may contain the
Plasmodium parasite or components thereof
(e.g., a target nucleic acid derived therefrom) - such as plasma, serum, lymph node, gastrointestinal
tissue, faeces, urine, semen or other body fluids or materials - is also contemplated.
The biological sample may be treated to physically or mechanically disrupt tissue
or cell structure, thus releasing intracellular components into a solution which may
further contain enzymes, buffers, salts, detergents and the like, which are used to
prepare, using standard methods, a biological sample for analysis. For example, a
sample may be treated with cell lysis reagent such as,
e.g., a lysis reagent as described in
US Pat. No. 10,093,989 or
PCT Pub. No. WO 2017/189746. Also, samples may include processed samples, such as those obtained from passing
samples over or through a filtering device, or following centrifugation, or by adherence
to a medium, matrix, or support.
[0066] "Nucleic acid" refers to a multimeric compound comprising two or more covalently
bonded nucleosides or nucleoside analogs having nitrogenous heterocyclic bases, or
base analogs, where the nucleosides are linked together by phosphodiester bonds or
other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric
DNA-RNA polymers or oligonucleotides, and analogs thereof. A nucleic acid "backbone"
may be made up of a variety of linkages, including one or more of sugar -phosphodiester
linkages, peptide-nucleic acid bonds (in "peptide nucleic acids" or PNAs,
see PCT Pub. No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof.
Sugar moieties of the nucleic acid may be either ribose or deoxyribose, or similar
compounds having known substitutions,
e.g., 2' methoxy substitutions and 2' halide substitutions
(e.g., 2'-F). Nitrogenous bases may be conventional bases (A, G, C, T, U), analogs thereof
(
e.g., inosine, 5-methylisocytosine, isoguanine;
The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992,
BioTechniques (2007) 43:617-24), which include derivatives of purine or pyrimidine bases (
e.g., N4-methyl deoxygaunosine, deaza- or aza-purines, deaza- or azapyrimidines, pyrimidine
bases having substituent groups at the 5 or 6 position, purine bases having an altered
or replacement substituent at the 2, 6 and/or 8 position, such as 2-amino-6-methylaminopurine,
06-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines,
and 04-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or 3-substituted
pyrazolo[3,4-d]pyrimidine;
US Pat. Nos. 5,378,825,
6,949,367 and
PCT Pub. No. WO 93/13121). Nucleic acids may include "abasic" residues in which the backbone does not include
a nitrogenous base for one or more residues (
US Pat. No. 5,585,481). A nucleic acid may comprise only conventional sugars, bases, and linkages as found
in RNA and DNA, or may include conventional components and substitutions (
e.g., conventional bases linked by a 2' methoxy backbone, or a nucleic acid including
a mixture of conventional bases and one or more base analogs). Nucleic acids may include
"locked nucleic acids" (LNA), in which one or more nucleotide monomers have a bicyclic
furanose unit locked in an RNA mimicking sugar conformation, which enhances hybridization
affinity toward complementary sequences in single-stranded RNA (ssRNA), single-stranded
DNA (ssDNA), or double-stranded DNA (dsDNA) (
Biochemistry (2004) 43:13233-41). Nucleic acids may include modified bases to alter the function or behavior of the
nucleic acid,
e.g., addition of a 3'-terminal dideoxynucleotide to block additional nucleotides from
being added to the nucleic acid. Synthetic methods for making nucleic acids in vitro
are well-known in the art.
[0067] The term "polynucleotide," as used herein, denotes a nucleic acid chain. Throughout
this application, nucleic acids are designated by the 5'-terminus to the 3'-terminus.
Standard nucleic acids,
e.g., DNA and RNA, are typically synthesized "5'-to-3',"
i.e., by the addition of nucleotides to the 3'-terminus of a growing nucleic acid.
[0068] A "nucleotide," as used herein, is a subunit of a nucleic acid consisting of a phosphate
group, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar found in RNA is
ribose. In DNA, the 5-carbon sugar is 2'-deoxyribose. The term also includes analogs
of such subunits, such as a methoxy group at the 2' position of the ribose (2'-0-Me).
[0069] A "nucleic-acid-based detection assay," as used herein, is an assay for the detection
of a target sequence within a target nucleic acid and utilizing one more oligonucleotides
that specifically hybridize to the target sequence.
[0070] In certain embodiments, a nucleic-acid-based detection assay is an "amplification-based
assay,"
i.e., an assay that utilizes one or more steps for amplifying a nucleic acid target sequence.
Various amplification methods for use in detection assays are known in the art, several
of which are summarized further herein. For the sake of clarity, an amplification-based
assay may include one or more steps that do not amplify a target sequence, such as,
for example, steps used in non-amplification-based assay methods (
e.g., a hybridization assay or a cleavage-based assay).
[0071] In other embodiments, a nucleic-acid-based detection assay is a "non-amplification-based
assay,"
i.e., an assay that does not rely on any step for amplifying a nucleic acid target sequence.
For the sake of clarity, a nucleic-acid-based detection assay that includes a reaction
for extension of a primer in the absence of any corresponding downstream amplification
oligomer (
e.g., extension of a primer by a reverse transcriptase to generate an RNA:DNA duplex
followed by an RNase digestion of the RNA, resulting in a single-stranded cDNA complementary
to an RNA target but without generating copies of the cDNA) is understood to be a
non-amplification-based assay.
[0072] An exemplary non-amplification-based assay is a "cleavage-based assay," which is
an assay that relies on the specific cleavage, by a flap endonuclease, of a linear
duplex cleavage structure formed by the specific hybridization of overlapping oligonucleotides
to a target nucleic acid. In these assays, a probe oligonucleotide containing a non-target-hybridizing
flap region is cleaved in an overlap-dependent manner by the flap endonuclease to
release a cleavage product that is then detected. The principles of cleavage-based
assays are well-known in the art, and exemplary assays are described in, for example,
Nat. Biotechnol. (1999) 17:292-296,
Mol. Diagn. (1999) 4: 135-144,
J. Clin. Microbiol. (2006) 44:3443-3447, and
US Patent Nos. 5,846,717,
6,706,471 and
5,614,402. Cleavage-based assays include,
e.g., the commercially available Invader
® assays (Hologic, Inc., Madison, WI).
[0073] A "target nucleic acid," as used herein, is a nucleic acid comprising a target sequence
to be detected. Target nucleic acids may be DNA or RNA as described herein, and may
be either single-stranded or double-stranded. The target nucleic acid may include
other sequences besides the target sequence.
[0074] By "isolated" it is meant that a sample containing a target nucleic acid is taken
from its natural milieu, but the term does not connote any degree of purification.
[0075] The term "target sequence," as used herein, refers to the particular nucleotide sequence
of a target nucleic acid that is to be detected. The "target sequence" includes the
complexing sequences to which oligonucleotides (
e.g., probe oligonucleotide, priming oligonucleotides and/or promoter oligonucleotides)
complex during a detection process (
e.g., an amplification-based detection assay such as, for example, TMA or PCR, or a non-amplification-based
detection assay such as, for example, a cleavage-based assay). Where the target nucleic
acid is originally single-stranded, the term "target sequence" will also refer to
the sequence complementary to the "target sequence" as present in the target nucleic
acid. Where the target nucleic acid is originally double-stranded, the term "target
sequence" refers to both the sense (+) and antisense (-) strands. In choosing a target
sequence, the skilled artisan will understand that a "unique" sequence should be chosen
so as to distinguish between unrelated or closely related target nucleic acids.
[0076] "Target-hybridizing sequence" is used herein to refer to the portion of an oligomer
that is configured to hybridize with a target nucleic acid sequence. Preferably, the
target-hybridizing sequences are configured to specifically hybridize with a target
nucleic acid sequence. Target-hybridizing sequences may be 100% complementary to the
portion of the target sequence to which they are configured to hybridize, but not
necessarily. Target-hybridizing sequences may also include inserted, deleted and/or
substituted nucleotide residues relative to a target sequence. Less than 100% complementarity
of a target-hybridizing sequence to a target sequence may arise, for example, when
the target nucleic acid is a plurality strains within a species, such as would be
the case for an oligomer configured to hybridize to the various strains
of Plasmodium. It is understood that other reasons exist for configuring a target-hybridizing sequence
to have less than 100% complementarity to a target nucleic acid.
[0077] The term "targets a sequence," as used herein in reference to a region of
Plasmodium sp. nucleic acid, refers to a process whereby an oligonucleotide hybridizes to the target
sequence in a manner that allows for detection as described herein. In one embodiment,
the oligonucleotide is complementary with the targeted
Plasmodium sp. nucleic acid sequence and contains no mismatches. In another embodiment, the oligonucleotide
is complementary but contains 1, 2, 3, 4, or 5 mismatches with the targeted
Plasmodium sp. nucleic acid sequence. Preferably, the oligonucleotide that hybridizes to the target
nucleic acid sequence includes at least 10 to as many as 50 nucleotides complementary
to the target sequence. It is understood that at least 10 and as many as 50 is an
inclusive range such that 10, 50 and each whole number there between are included.
Preferably, the oligomer specifically hybridizes to the target sequence.
[0078] The term "configured to" denotes an actual arrangement of the polynucleotide sequence
configuration of a referenced oligonucleotide target-hybridizing sequence. For example,
oligonucleotides that are configured to specifically hybridize to a target sequence
have a polynucleotide sequence that specifically hybridizes to the referenced sequence
under stringent hybridization conditions.
[0079] The term "configured to specifically hybridize to" as used herein means that the
target-hybridizing region of an oligonucleotide is designed to have a polynucleotide
sequence that could target a sequence of the referenced
Plasmodium sp. target region. Such an oligonucleotide is not limited to targeting that sequence
only, but is rather useful as a composition, in a kit or in a method for targeting
a
Plasmodium sp. target nucleic acid. The oligonucleotide is designed to function as a component of
an assay for detection
of Plasmodium sp. from a sample, and therefore is designed to target
Plasmodium sp. in the presence of other nucleic acids commonly found in testing samples. "Specifically
hybridize to" does not mean exclusively hybridize to, as some small level of hybridization
to non-target nucleic acids may occur, as is understood in the art. Rather, "specifically
hybridize to" means that the oligonucleotide is configured to function in an assay
to primarily hybridize the target so that an accurate detection of target nucleic
acid in a sample can be determined. The term "configured to" denotes an actual arrangement
of the polynucleotide sequence configuration of the oligonucleotide target-hybridizing
sequence.
[0080] The term "fragment," as used herein in reference to a
Plasmodium sp. targeted nucleic acid, refers to a piece of contiguous nucleic acid.
[0081] The term "region," as used herein, refers to a portion of a nucleic acid wherein
the portion is smaller than the entire nucleic acid. For example, when the nucleic
acid in reference is an oligonucleotide promoter primer, the term "region" may be
used refer to the smaller promoter portion of the entire oligonucleotide. As a non-limiting
example, when the nucleic acid in reference is an amplicon, the term region may be
used to refer to the smaller nucleotide sequence identified for hybridization by the
target-hybridizing sequence of a probe.
[0082] The interchangeable terms "oligomer," "oligo," and "oligonucleotide" refer to a nucleic
acid having generally less than 1,000 nucleotide (nt) residues, including polymers
in a range having a lower limit of about 5 nt residues and an upper limit of about
500 to 900 nt residues. In some embodiments, oligonucleotides are in a size range
having a lower limit of about 12 to 15 nt and an upper limit of about 50 to 600 nt,
and other embodiments are in a range having a lower limit of about 15 to 20 nt and
an upper limit of about 22 to 100 nt. Oligonucleotides may be purified from naturally
occurring sources or may be synthesized using any of a variety of well-known enzymatic
or chemical methods. The term oligonucleotide does not denote any particular function
to the reagent; rather, it is used generically to cover all such reagents described
herein. An oligonucleotide may serve various different functions. For example, it
may function as a primer if it is specific for and capable of hybridizing to a complementary
strand and can further be extended in the presence of a nucleic acid polymerase; it
may function as a primer and provide a promoter if it contains a sequence recognized
by an RNA polymerase and allows for transcription (
e.g., a T7 Primer); and it may function to detect a target nucleic acid if it is capable
of hybridizing to the target nucleic acid, or an amplicon thereof, and further provides
a detectible moiety (
e.g., an acridinium-ester compound).
[0083] As used herein, an oligonucleotide can "substantially correspond to" a specified
reference nucleic acid sequence, which means that the oligonucleotide is sufficiently
similar to the reference nucleic acid sequence such that the oligonucleotide has similar
hybridization properties to the reference nucleic acid sequence in that it would hybridize
with the same target nucleic acid sequence under stringent hybridization conditions.
One skilled in the art will understand that "substantially corresponding oligonucleotides"
can vary from a reference sequence and still hybridize to the same target nucleic
acid sequence. It is also understood that a first nucleic acid corresponding to a
second nucleic acid includes the RNA and DNA thereof and includes the complements
thereof, unless the context clearly dictates otherwise. This variation from the nucleic
acid may be stated in terms of a percentage of identical bases within the sequence
or the percentage of perfectly complementary bases between the probe or primer and
its target sequence. Thus, in certain embodiments, an oligonucleotide "substantially
corresponds" to a reference nucleic acid sequence if these percentages of base identity
or complementarity are from 100% to about 80%. In preferred embodiments, the percentage
is from 100% to about 85%. In more preferred embodiments, this percentage is from
100% to about 90%; in other preferred embodiments, this percentage is from 100% to
about 95%. Similarly, a region of a nucleic acid or amplified nucleic acid can be
referred to herein as corresponding to a reference nucleic acid sequence. One skilled
in the art will understand the various modifications to the hybridization conditions
that might be required at various percentages of complementarity to allow hybridization
to a specific target sequence without causing an unacceptable level of non-specific
hybridization.
[0084] Exemplary sequences for
Plasmodium sp. target nucleic acid are shown in Table 19,
infra. Specifically, SEQ ID NOs:180 and 192-195 are reference sequences corresponding to
ribosomal RNA sequences for
Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale, and
Plasmodium malariae, respectively. Where a target region of
Plasmodium sp. is described herein as "corresponding to" a defined region of SEQ ID NO: 180,
it is understood that such reference includes homologous regions of any one or more
of SEQ ID NOs: 192-195. It is also understood that such reference to a region "corresponding
to" a defined region of SEQ ID NO: 180 includes homologous regions of naturally occurring
variants of any one or more of SEQ ID NOs: 180 and 192-195 that may be present in
a sample.
[0085] An "amplification oligomer" is an oligomer at least the 3'-end of which is complementary
to a target nucleic acid and which hybridizes to a target nucleic acid, or its complement,
and participates in a nucleic acid amplification reaction. An example of an amplification
oligomer is a "primer" that hybridizes to a target nucleic acid and contains a 3'
OH end that is extended by a polymerase in an amplification process. Another example
of an amplification oligomer is an oligomer that is not extended by a polymerase (
e.g., because it has a 3' blocked end) but participates in or facilitates amplification.
For example, the 5' region of an amplification oligonucleotide may include a promoter
sequence that is non-complementary to the target nucleic acid (which may be referred
to as a "promoter primer" or "promoter provider"). Those skilled in the art will understand
that an amplification oligomer that functions as a primer may be modified to include
a 5' promoter sequence, and thus function as a promoter primer. Incorporating a 3'
blocked end further modifies the promoter primer, which is now capable of hybridizing
to a target nucleic acid and providing an upstream promoter sequence that serves to
initiate transcription, but does not provide a primer for oligo extension. Such a
modified oligo is referred to herein as a "promoter provider" oligomer. Size ranges
for amplification oligonucleotides include those that are about 10 to about 70 nt
long (not including any promoter sequence or poly-A tails) and contain at least about
10 contiguous bases, or even at least 12 contiguous bases that are complementary to
a region of the target nucleic acid sequence (or a complementary strand thereof).
The contiguous bases are at least 80%, or at least 90%, or completely complementary
to the target sequence to which the amplification oligomer binds. An amplification
oligomer may optionally include modified nucleotides or analogs, or additional nucleotides
that participate in an amplification reaction but are not complementary to or contained
in the target nucleic acid, or template sequence. It is understood that when referring
to ranges for the length of an oligonucleotide, amplicon, or other nucleic acid, that
the range is inclusive of all whole numbers (
e.g., 19-25 contiguous nucleotides in length includes 19, 20, 21, 22, 23, 24 & 25).
[0086] As used herein, a "promoter" is a specific nucleic acid sequence that is recognized
by a DNA-dependent RNA polymerase ("transcriptase") as a signal to bind to the nucleic
acid and begin the transcription of RNA at a specific site.
[0087] As used herein, a "promoter provider" or "provider" refers to an oligonucleotide
comprising first and second regions and which is modified to prevent the initiation
of DNA synthesis from its 3' -terminus. The "first region" of a promoter provider
oligonucleotide comprises a base sequence which hybridizes to a DNA template, where
the hybridizing sequence is situated 3', but not necessarily adjacent to, a promoter
region. The hybridizing portion of a promoter oligonucleotide is typically at least
10 nucleotides in length and may extend up to 50 or more nucleotides in length. The
"second region" comprises a promoter sequence for an RNA polymerase. A promoter oligonucleotide
is engineered so that it is incapable of being extended by an RNA- or DNA-dependent
DNA polymerase,
e.g., reverse transcriptase, preferably comprising a blocking moiety at its 3'-terminus
as described above. As referred to herein, a "T7 Provider" is a blocked promoter provider
oligonucleotide that provides an oligonucleotide sequence that is recognized by T7
RNA polymerase.
[0088] "Amplification" refers to any known procedure for obtaining multiple copies of a
target nucleic acid sequence or its complement or fragments thereof. The multiple
copies may be referred to as amplicons or amplification products. Known amplification
methods include both thermal cycling and isothermal amplification methods. In some
embodiments, isothermal amplification methods are preferred. Replicase-mediated amplification,
polymerase chain reaction (PCR), ligase chain reaction (LCR), strand-displacement
amplification (SDA), and transcription-mediated or transcription-associated amplification
are non-limiting examples of nucleic acid amplification methods. Replicase-mediated
amplification uses self-replicating RNA molecules, and a replicase such as QB-replicase
(
e.g., US Pat. No. 4,786,600). PCR amplification uses a DNA polymerase, pairs of primers, and thermal cycling
to synthesize multiple copies of two complementary strands of dsDNA or from a cDNA
(e.g., US Pat. Nos. 4,683,195,
4,683,202, and
4,800, 159). LCR amplification uses four or more different oligonucleotides to amplify a target
and its complementary strand by using multiple cycles of hybridization, ligation,
and denaturation (
e.g.,
US Pat. No. 5,427,930 and
US Pat. No. 5,516,663). SDA uses a primer that contains a recognition site for a restriction endonuclease
and an endonuclease that nicks one strand of a hemimodified DNA duplex that includes
the target sequence, whereby amplification occurs in a series of primer extension
and strand displacement steps (
e.g.,
US Pat. No. 5,422,252;
US Pat. No. 5,547,861; and
US Pat. No. 5,648,211). Preferred embodiments use an amplification method suitable for the amplification
of RNA target nucleic acids, such as transcription-mediated amplification (TMA) or
NASBA, but it will be apparent to persons of ordinary skill in the art that oligomers
disclosed herein may be readily used as primers in other amplification methods.
[0089] "Transcription-associated amplification," also referred to herein as "transcription-mediated
amplification" (TMA), refers to nucleic acid amplification that uses an RNA polymerase
to produce multiple RNA transcripts from a nucleic acid template. These methods generally
employ an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside
triphosphates, and a template complementary oligonucleotide that includes a promoter
sequence, and optionally may include one or more other oligonucleotides. TMA methods
are embodiments of amplification methods used for amplifying and detecting
Plasmodium target sequences as described herein. Variations of transcription-associated amplification
are well-known in the art as previously disclosed in detail (
e.g.,
US Pat. Nos. 4,868,105;
5,124,246;
5,130,238;
5,437,990;
5,554,516; and
7,374,885; and
PCT Pub. Nos. WO 88/01302,
WO 88/10315, and
WO 95/03430). The person of ordinary skill in the art will appreciate that the disclosed compositions
may be used in amplification methods based on extension of oligomer sequences by a
polymerase.
[0090] As used herein, the term "real-time TMA" refers to transcription-mediated amplification
("TMA") of target nucleic acid that is monitored by real-time detection means.
[0091] The term "amplicon," which is used interchangeably with "amplification product,"
refers to the nucleic acid molecule generated during an amplification procedure that
is complementary or homologous to a sequence contained within the target sequence.
These terms can be used to refer to a single strand amplification product, a double
strand amplification product, or one of the strands of a double strand amplification
product.
[0092] "Probe," "detection probe," "detection oligonucleotide," and "detection probe oligomer"
are used interchangeably herein to refer to a nucleic acid oligomer that hybridizes
specifically to a target sequence in a nucleic acid, or in an amplified nucleic acid,
under conditions that promote hybridization to allow detection of the target sequence
or amplified nucleic acid. Detection may either be direct
(e.g., a probe hybridized directly to its target sequence) or indirect
(e.g., a probe linked to its target via an intermediate molecular structure). Probes may
be DNA, RNA, analogs thereof, or combinations thereof and they may be labeled or unlabeled.
A probe's "target sequence" generally refers to a smaller nucleic acid sequence within
a larger nucleic acid sequence that hybridizes specifically to at least a portion
of a probe oligomer by standard base pairing. A probe may comprise target-specific
sequences and other sequences that contribute to the three-dimensional conformation
of the probe
(e.g., US Pat. Nos. 5,118,801;
5,312,728;
6,849,412;
6,835,542;
6,534,274; and
6,361,945; and
US Pub. No. 20060068417). In a preferred embodiment, the detection probe comprises a 2' methoxy backbone,
which can result in a higher signal being obtained.
[0093] The term "TaqMan
® probe" refers to detection oligonucleotides that contain a fluorescent dye, typically
on the 5' base, and a non-fluorescent quenching dye (quencher), typically on the 3'
base. When irradiated, the excited fluorescent dye transfers energy to the nearby
quenching dye molecule rather than fluorescing, resulting in a non-fluorescent substrate.
During amplification, the exonuclease activity of the polymerase cleaves the TaqMan
probe to separate the fluorophore from the quencher, thereby allowing an unquenched
signal to be emitted from the fluorophore as an indicator of amplification.
[0094] As used herein, a "label" refers to a moiety or compound joined directly or indirectly
to a probe that is detected or leads to a detectable signal. Direct labeling can occur
through bonds or interactions that link the label to the probe, including covalent
bonds or non-covalent interactions,
e.g., hydrogen bonds, hydrophobic and ionic interactions, or formation of chelates or
coordination complexes. Indirect labeling can occur through use of a bridging moiety
or "linker" such as a binding pair member, an antibody or additional oligomer, which
is either directly or indirectly labeled, and which may amplify the detectable signal.
Labels include any detectable moiety, such as a radionuclide, ligand (
e.g., biotin, avidin), enzyme or enzyme substrate, reactive group, or chromophore (
e.g., dye, particle, or bead that imparts detectable color), luminescent compound (
e.g., bioluminescent, phosphorescent, or chemiluminescent labels), or fluorophore. Labels
may be detectable in a homogeneous assay in which bound labeled probe in a mixture
exhibits a detectable change different from that of an unbound labeled probe,
e.g., instability or differential degradation properties. A "homogeneous detectable label"
can be detected without physically removing bound from unbound forms of the label
or labeled probe
(e.g., US Pat. Nos. 5,283, 174, 5,656,207, and
5,658,737). Labels include chemiluminescent compounds,
e.g., acridinium ester ("AE") compounds that include standard AE and derivatives (
e.g.,
US Pat. Nos. 5,656,207,
5,658,737, and
5,639,604). Synthesis and methods of attaching labels to nucleic acids and detecting labels
are well known
(e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10;
US Pat. Nos. 5,658,737,
5,656,207,
5,547,842,
5,283,174, and
4,581,333). More than one label, and more than one type of label, may be present on a particular
probe, or detection may use a mixture of probes in which each probe is labeled with
a compound that produces a detectable signal
(e.g., US Pat. Nos. 6, 180,340 and
6,350,579).
[0095] As used herein, structures referred to as "molecular torches" are designed to include
distinct regions of self-complementarity ("the closing domain") which are connected
by a joining region ("the target-binding domain") and which hybridize to one another
under predetermined hybridization assay conditions. All or part of the nucleotide
sequences comprising closing domains may also function as target-binding domains.
Thus, closing domains can include target-binding sequences, non-target binding sequences,
and combinations thereof.
[0096] As used herein, structures referred to as "molecular beacons" are designed to include
a target-binding sequence flanked on both its 5' and 3' ends by sequences that are
complementary to each other and which hybridize to each other under predetermined
hybridization assay conditions. The flanking, complementary regions may be referred
to as "switch sequences."
[0097] "Capture probe," "capture oligonucleotide," "target capture oligonucleotide," and
"capture probe oligomer" are used interchangeably herein to refer to a nucleic acid
oligomer that specifically hybridizes to a target sequence in a target nucleic acid
by standard base pairing and joins to a binding partner on an immobilized probe to
capture the target nucleic acid to a support. One example of a capture oligomer includes
an oligonucleotide comprising two binding regions: a target hybridizing sequence and
an immobilized probe-binding region. A variation of this example, the two regions
may be present on two different oligomers joined together by one or more linkers.
In another embodiment of a capture oligomer, the target-hybridizing sequence is a
sequence that includes random or non-random poly-GU, poly-GT, or poly U sequences
to bind non-specifically to a target nucleic acid and link it to an immobilized probe
on a support
(see, e.g., PCT Pub. No. WO 2008/016988). The immobilized probe-binding region can be a nucleic acid sequence, referred to
as a tail. Tails include a substantially homopolymeric tail of about 10 to 40 nucleotides
(
e.g., A10 to A40), or of about 17 to 33 nt
(e.g., T3A14 to T3A30), that bind to a complementary immobilized sequence attached to the
support particle or support matrix. Thus, a non-limiting example of preferred nucleic
acid tails can in some embodiments include T
0-4A
10-40 sequences. Another example of a capture oligomer comprises two regions, a target-hybridizing
sequence and a binding pair member that is not a nucleic acid sequence.
[0098] As used herein, an "immobilized oligonucleotide," "immobilized probe," or "immobilized
nucleic acid" refers to a nucleic acid binding partner that joins a capture oligomer
to a support, directly or indirectly. An immobilized probe joined to a support facilitates
separation of a capture probe bound target from unbound material in a sample. One
embodiment of an immobilized probe is an oligomer joined to a support that facilitates
separation of bound target sequence from unbound material in a sample. Supports may
include known materials, such as matrices and particles free in solution, which may
be made of nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,
silane, polypropylene, metal, or other compositions, of which one embodiment is magnetically
attractable particles. Supports may be monodisperse magnetic spheres (
e.g., uniform size + 5%), to which an immobilized probe is joined directly (via covalent
linkage, chelation, or ionic interaction), or indirectly (via one or more linkers),
where the linkage or interaction between the probe and support is stable during hybridization
conditions.
DESCRIPTION
[0099] The present invention is generally directed to methods and compositions for determining
the presence or absence of the protozoan parasite
Plasmodium sp. in a sample, such as,
e.g., a blood sample. Suitably, the methods and compositions described herein are able
to detect the presence or absence of
Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and/or
Plasmodium vivax. In some embodiments, the present invention provides methods for the detection of
Plasmodium sp. in a sample, where the method includes performing amplification-based detection
of a target nucleic from
Plasmodium sp. The present invention further provides compositions (including reaction mixtures)
and kits comprising a combination of oligomers for detecting
Plasmodium sp. - including
Plasmodium falciparum and/or
Plasmodium knowlesi and/or
Plasmodium malariae and/or
Plasmodium ovale and/or
Plasmodium vivax - in a sample. The oligomer combination generally includes at least two amplification
oligomers for detecting
Plasmodium sp. - including
Plasmodium falciparum and/or
Plasmodium knowlesi and/or
Plasmodium malariae and/or
Plasmodium ovale and/or
Plasmodium vivax - in a sample, and may further include one or more additional oligomers as described
herein for performing amplification-based detection of
Plasmodium sp. - including
Plasmodium falciparum and/or
Plasmodium knowlesi and/or
Plasmodium malariae and/or
Plasmodium ovale and/or
Plasmodium vivax - such as, for example, a capture probe and/or a detection probe.
[0100] Methods for detecting the presence or absence of
Plasmodium sp. in a sample from a subject generally include performing a nucleic-acid-based
detection assay for the specific detection in the sample of
Plasmodium sp. nucleic acid. Nucleic-acid-based detection assays generally utilize oligonucleotides
that specifically hybridize to a target nucleic acid of
Plasmodium sp. with minimal cross-reactivity to other nucleic acids suspected of being in a
sample. In some variations, an oligonucleotide or combination of oligonucleotides
for nucleic-acid-based detection of
Plasmodium sp. has minimal cross-reactivity to
Babesia sp. (
e.g., B. microti) nucleic acids.
[0101] In certain aspects of the invention, a combination of at least two oligomers is provided
for determining the presence or absence of
Plasmodium species in a sample. Typically, the oligomer combination includes at least first
and second amplification oligomers for amplifying a
Plasmodium sp. target region corresponding to a region of SEQ ID NO:180. In such embodiments,
at least one amplification oligomer comprises a target-hybridizing sequence in the
sense orientation ("sense THS") and at least one amplification oligomer comprises
a target-hybridizing sequence in the antisense orientation ("antisense THS"), where
the sense THS and antisense THS of the amplification oligomers are each configured
to specifically hybridize to a
Plasmodium sp. target sequence corresponding to a sequence contained within SEQ ID NO: 180,
and where the target-hybridizing sequences are selected such that the
Plasmodium sequence targeted by the antisense THS is situated downstream of the
Plasmodium sequence targeted by the sense THS (
i.e., the at least two amplification oligomers are situated such that they flank the target
region to be amplified).
[0102] In some embodiments, the
Plasmodium sp. target region corresponds to a region of SEQ ID NO:180 from about nucleotide
position 844 or about nucleotide position 910 to about nucleotide position 1038, about
nucleotide position 1051, about nucleotide position 1060, or about nucleotide position
1077. In other embodiments, the
Plasmodium sp. target region corresponds to a region of SEQ ID NO: 180 from about nucleotide
position 1153, about nucleotide position 1169, or about nucleotide position 1182 to
about nucleotide position 1327, about nucleotide position 1354, or about nucleotide
position 1382.
[0103] In some embodiments, a composition includes an amplification oligomer comprising
a
Plasmodium-specific target-hybridizing sequence substantially corresponding to, or identical to, the
sequence shown in any one of SEQ ID NOs:21-56, 80-102, and 182-184. In such variations,
the oligomer combination includes at least one amplification oligomer comprising
a Plasmodium-specific target-hybridizing sequence of the opposite polarity (sense vs. antisense or
vice versa) as the target-hybridizing sequence of the oligomer as above, such that at least
two amplification oligomers flank a target region to be amplified.
[0104] In some embodiments, a composition includes (1) at least one amplification oligomer
comprising a
Plasmodium-specific target-hybridizing region substantially corresponding to at least one sense oligomer
sequence depicted in Table 1 below, and (2) at least one amplification oligomer comprising
a
Plasmodium-specific target hybridizing region substantially corresponding to at least one antisense oligomer
sequence depicted in Table 1. In particular variations, the sense and/or antisense
target-hybridizing sequence(s) of an amplification oligomer combination comprises
or consists of the sense and/or antisense sequence(s) selected from Table 1.
Table 1. Exemplary Sense and Antisense Amplification Oligomer Target-hybridizing Sequences
for Amplification
of Plasmodium species Target Regions
SEQ ID NO |
Sequence (5' → 3') |
Sense/Antisense1 |
21 |
AATACTACAGCATGG |
Sense |
22 |
GGAAGGCAGCAGGCGCGTA |
Sense |
23 |
AATACTACAGCATGGA |
Sense |
24 |
AATACTACAGCATGGAA |
Sense |
25 |
ATACTACAGCATGGAATA |
Sense |
26 |
ATTCAGATGTCAGAGGTGA |
Sense |
27 |
GTATTCAGATGTCAGAGGTGA |
Sense |
28 |
GTTACGATTAATAGGAGT |
Sense |
29 |
GTTACGATTAATAGGAGTA |
Sense |
30 |
GTTACGATTAATAGGAGTAG |
Sense |
31 |
GTTACGATTAATAGGAGTAGC |
Sense |
32 |
AATACTACAGCATGGAAT |
Sense |
33 |
AATACTACAGCATGGAATA |
Sense |
34 |
TACGATTAATAGGAGT |
Sense |
35 |
TACTACAGCATGGAATA |
Sense |
36 |
TATTCAGATGTCAGAGGTGA |
Sense |
37 |
TCAGTNCCTTATGAGAAATC |
Sense |
38 |
TGGCTTAGTTACGATT |
Sense |
39 |
TGGCTTAGTTACGATTAATAG |
Sense |
40 |
TTAATAGGAGTAGCTTGGGG |
Sense |
41 |
TTACGATTAATAGGAGT |
Sense |
42 |
TTCAGATGTCAGAGGTGA |
Sense |
43 |
TTGGCTTAGTTACGAT |
Sense |
44 |
TTGGCTTAGTTACGATTA |
Sense |
45 |
TTGGGGACATTCGTATTCAGA |
Sense |
46 |
TTTAGATTGCTTCCTTCAGT |
Sense |
47 |
TTTGAATACTANAGCA |
Sense |
48 |
ACATTCGTATTCAGATGTCAG |
Sense |
49 |
CTTAGTTACGATTAATAGGA |
Sense |
50 |
CGATTAATAGGAGTAGCTTGG |
Sense |
51 |
CTTAGTTACGATTAATAGGAGTAG |
Sense |
52 |
CTTGAATACTNCAGCA |
Sense |
53 |
GGCTTAGTTACGATTA |
Sense |
54 |
AATACTANAGCATGG |
Sense |
55 |
AATACTANAGCATGGAATA |
Sense |
56 |
AATTCTAAAGAAGAGAG |
Sense |
80 |
TTCACTCCCTTAACTTTCGTTCTTG |
Antisense |
81 |
CTTGATTAATGGAAGTATTTTAGA |
Antisense |
82 |
CTTAACTTTCGTTCTTGATTAATGGAAGT |
Antisense |
83 |
CCTACTCTTGTCTTAAACTA |
Antisense |
84 |
AAACGGCCATGCATCACCATCCAAGA |
Antisense |
85 |
CTCCCTTAACTTTCGTTCTTGATTAATGGAAGT |
Antisense |
86 |
CGACGGTATCTGATCGTCTTCACTCCC |
Antisense |
87 |
CTTAACTTTCGTTCTTGATTAATGGAAG |
Antisense |
88 |
CTTAACTTTCGTTCTTGATTAATGGAAGTA |
Antisense |
89 |
CACTCCCTTAACTTTCGTTCTTGATTAATG |
Antisense |
90 |
CACTCCCTTAACTTTCGTTCTTGATTAATGG |
Antisense |
91 |
CTTCACTCCCTTAACTTTCGTTCTTGATT |
Antisense |
92 |
CTTCACTCCCTTAACTTTCGTTCTTGAT |
Antisense |
93 |
ATCGTCTTCACTCCCTTAACTTTCGTTC |
Antisense |
94 |
CTCCCTTAACTTTCGTTCTTGATTAATG |
Antisense |
95 |
TCACTCCCTTAACTTTCGTTCTTGAT |
Antisense |
96 |
CCCTTAACTTTCGTTCTTGATTAATG |
Antisense |
97 |
CTTAACTTTCGTTCTTGATTAATG |
Antisense |
98 |
TAACTTTCGTTCTTGATTAATG |
Antisense |
99 |
ACTCCCTTAACTTTCGTTCTTGAT |
Antisense |
100 |
TCCCTTAACTTTCGTTCTTGAT |
Antisense |
101 |
AGGCAAATGCTTTCGCAGTTGTTNGTCT |
Antisense |
102 |
AGGCAAATGCTTTCGCAGTTGTTTGTCT |
Antisense |
182 |
TCAAGAAAGAGCTATNAATCTGTCAATCC |
Antisense |
183 |
GAAATCAAAGTCTTTGGGTTCTG |
Sense |
184 |
CAAAGTCTTTGGGTTCTGG |
Sense |
1The Sense/Antisense designation of these sequences is for exemplary purposes only.
Such designation does not necessarily limit a sequence to the accompanying designation. |
[0105] In some embodiments, an oligomer combination comprises (a) an amplification oligomer
comprising a target-hybridizing sequence (i) that is from about 14 to about 20 contiguous
nucleotides in length, is contained in the sequence of SEQ ID NO:162, and includes
the sequence of SEQ ID NO:163, or (ii) that is from about 14 to about 25 contiguous
nucleotides in length, is contained in the sequence of SEQ ID NO: 166, and includes
SEQ ID NO: 167 or SEQ ID NO:168; and (b) an amplification oligomer comprising a target-hybridizing
sequence that is from about 15 to about 33 contiguous nucleotides in length, is contained
in SEQ ID NO:169 and includes the sequence of SEQ ID NO:171, SEQ ID NO:172, or SEQ
ID NO:173. In some such embodiments, the oligomer combination comprises an amplification
oligomer of (a)(i) where the target-hybridizing sequence of is selected from SEQ ID
NOs:21, 23-25, 32, 33, 35, 54, and 55, or where the target-hybridizing sequence is
contained in the sequence of SEQ ID NO: 164 and includes the sequence of SEQ ID NO:165
(e.g., a target-hybridizing sequence selected from SEQ ID NOs:21, 23-25, 32, 33, and 35).
In yet other embodiments, the oligomer combination comprises an amplification oligomer
of (a)(ii) where the target-hybridizing sequence of includes the sequence of SEQ ID
NO:167
(e.g., a target-hybridizing sequence selected from the SEQ ID NOs:28-31, 34, 40, 41, and
49-51), or where the target-hybridizing sequence includes the sequence of SEQ ID NO:
168
(e.g., a target-hybridizing sequence selected from SEQ ID NOs:38, 39, 43, 44, and 53). In
certain embodiments of an oligomer combination as above, the target-hybridizing sequence
of (b) is selected from SEQ ID NOs:80-82 and 85-100. In other embodiments, the target-hybridizing
sequence of (b) is contained in the sequence of SEQ ID NO:170 and includes the sequence
of SEQ ID NO:171
(e.g., a target-hybridizing sequence selected from SEQ ID NOs:81, 82, 85, 87-90, 94, and
96-98), or is contained in the sequence of SEQ ID NO:170 and includes the sequence
of SEQ ID NO: 172
(e.g., a target-hybridizing sequence selected from SEQ ID NOs:80, 82, 85, and 87-100).
[0106] In some embodiments, an oligomer combination comprises (a') an amplification oligomer
comprising a target-hybridizing sequence that is contained in the sequence of SEQ
ID NO:185 and includes the sequence of SEQ ID NO:37, SEQ ID NO:46, or SEQ ID NO:187;
and (b') an amplification oligomer comprising a target-hybridizing sequence that is
contained in the sequence of SEQ ID NO:188 and includes the sequence of SEQ ID NO:83,
SEQ ID NO:84, or SEQ ID NO:182. In some embodiments, the amplification oligomer of
(a') comprises a target-hybridizing sequence selected from SEQ ID NOs:37, 46, 183,
and 184, or a target-hybridizing sequence contained in the sequence of SEQ ID NO:186
(e.g., a target-hybridizing sequence of SEQ ID NO: 183 or SEQ ID NO: 184). In certain embodiments,
the amplification oligomers of (b') comprises a target-hybridizing sequence selected
from SEQ ID NOs:83, 84, and 182.
[0107] In certain embodiments, an amplification oligomer as described herein is a promoter
primer or promoter provider further comprising a promoter sequence located 5' to the
target-hybridizing sequence and which is non-complementary to the
Plasmodium sp. target nucleic acid. For example, in some embodiments of an oligomer combination
as described herein, an amplification oligomer of (b) or (b') as described above is
a promoter primer further comprising a 5' promoter sequence. In particular embodiments,
the promoter sequence is a T7 RNA polymerase promoter sequence such as, for example,
a T7 promoter sequence having the sequence shown in SEQ ID NO: 179. In specific variations,
an amplification oligomer is a promoter primer having the sequence shown selected
from SEQ ID NOs:57-77 and 181.
[0108] Table 2 shows particularly suitable combinations of amplification oligomer target-hybridizing
sequences ("Amp 1" and "Amp 2") for detection of
Plasmodium species target nucleic acid.
Table 2. Exemplary Combinations of Amplification Oligomer Target-hybridizing Sequences.
Amp 1 (SEQ ID NO) |
Amp 2 (SEQ ID NO) |
30 |
5 |
33 |
8 |
49 |
11 |
21 |
14 |
30 |
17 |
33 |
20 |
49 |
23 |
21 |
26 |
30 |
29 |
21 |
32 |
34 |
35 |
53 |
38 |
21 |
41 |
34 |
44 |
53 |
46 |
183 |
182 |
184 |
182 |
[0109] In some embodiments, an oligomer combination as above includes at least two sense
amplification oligomers and/or at least two antisense amplification oligomers flanking
a
Plasmodium sp. target region. For example, an oligomer combination may include (a) at least
two amplification oligomer (
e.g., two or three amplification oligomers) each comprising a target-hybridizing sequence
that is from about 14 to about 25 contiguous nucleotides in length, is contained in
the sequence of SEQ ID NO: 166, and includes SEQ ID NO: 167 or SEQ ID NO: 16 and/or
(b) at least two amplification oligomers (
e.g., two or three amplification oligomers) each comprising a target-hybridizing sequence
that is from about 15 to about 33 contiguous nucleotides in length, is contained in
SEQ ID NO: 169 and includes the sequence of SEQ ID NO:171, SEQ ID NO: 172, or SEQ
ID NO:173; in some such variations, the oligomer combination includes (a) a first
amplification oligomer comprising a target-hybridizing sequence that includes the
sequence of SEQ ID NO: 167
(e.g., the target-hybridizing sequence of SEQ ID NO:34) and a second amplification oligomer
comprising a target-hybridizing sequence that includes the sequence of SEQ ID NO:
168
(e.g., the target-hybridizing sequence of SEQ ID NO:53). In other embodiments comprising
at least two sense amplification oligomers and/or at least two antisense amplification
oligomers, an oligomer combination comprises (a)(i) an amplification oligomer comprising
a target-hybridizing sequence that is from about 14 to about 20 contiguous nucleotides
in length, is contained in the sequence of SEQ ID NO:162, and includes the sequence
of SEQ ID NO:163, and (ii) an amplification oligomer comprising a target-hybridizing
sequence that is from about 14 to about 25 contiguous nucleotides in length, is contained
in the sequence of SEQ ID NO: 166, and includes SEQ ID NO: 167 or SEQ ID NO: 168;
and/or (b) at least two amplification oligomers (
e.g., two or three amplification oligomers) each comprising a target-hybridizing sequence
that is from about 15 to about 33 contiguous nucleotides in length, is contained in
SEQ ID NO: 169 and includes the sequence of SEQ ID NO: 171, SEQ ID NO: 172, or SEQ
ID NO: 173; in some such variations, the oligomer combination includes (a)(i) an amplification
oligomer comprising a target-hybridizing sequence that is contained in the sequence
of SEQ ID NO: 164 and includes the sequence of SEQ ID NO: 165
(e.g., the target-hybridizing sequence of SEQ ID NO:21), and (a)(ii) an amplification comprising
a target-hybridizing sequence that includes the sequence of SEQ ID NO: 167
(e.g., the target-hybridizing sequence of SEQ ID NO:34). In some embodiments of an oligomer
combination as above comprising at least two amplification oligomers of (b), oligomer
combination comprise first and second amplification oligomers of (b), each comprising
a target-hybridizing sequence that is contained in SEQ ID NO:170 and includes the
sequence of SEQ ID NO:171 or SEQ ID NO: 172
(e.g., a first amplification oligomer comprising the target-hybridizing sequence of SEQ
ID NO:94 and a second amplification oligomer comprising the target-hybridizing sequence
of SEQ ID NO:95).
[0110] In some embodiments, an oligomer combination as described herein further comprises
at least one capture probe oligomer comprising a target-hybridizing sequence configured
to specifically hybridize to
Plasmodium species target nucleic acid. In some such embodiments, the capture probe oligomer
comprises a target-hybridizing sequence a sequence substantially corresponding to
to a sequence contained in the complement of SEQ ID NO:180. In some embodiments, a
capture probe oligomer target-hybridizing sequence is covalently attached to a sequence
or moiety that binds to an immobilized probe. Suitable capture probe oligomer target-hybridizing
sequences include sequences that are up to about 30 contiguous nucleotides in length
and include a sequence substantially corresponding to a sequence selected from SEQ
ID NOs:11-15, 17, 19, and 20
(e.g., a target-hybridizing sequence comprising or consisting of a sequence selected from
SEQ ID NOs:11-15, 17, 19, and 20, including DNA equivalents and DNA/RNA chimerics
thereof). In some embodiments, a capture probe oligomer comprises or consists of a
sequence selected from SEQ ID NOs: 1-5, 7, 9, and 10. In some embodiments, the oligomer
combination includes at least two capture probe oligomers
(e.g., at least two capture probe oligomers as above). A first capture probe oligomer comprising
the target-hybridizing sequence of SEQ ID NO:19
(e.g., a capture probe oligomer comprising the sequence of SEQ ID NO:9) and a second capture
probe oligomer comprising the target-hybridizing sequence of SEQ ID NO:20
(e.g., a capture probe oligomer comprising the sequence of SEQ ID NO:10) are particularly
suitable for use together in oligomer combinations as described herein.
[0111] In certain variations, an oligomer combination as described herein further comprises
at least one detection probe oligomer configured to specifically hybridize to a
Plasmodium sp. target sequence that is amplifiable using the at least two amplification oligomers
targeting a
Plasmodium sp. target region. In some embodiments where a
Plasmodium sp. target region corresponds to a region of SEQ ID NO:180 from about nucleotide
position 844 or about nucleotide position 910 to about nucleotide position 1038, about
nucleotide position 1051, about nucleotide position 1060, or about nucleotide position
1077, the oligomer combination includes a detection probe oligomer that specifically
hybridizes to a target region corresponding to a region of SEQ ID NO:180 from about
nucleotide position 951 to about nucleotide position 998 or the full complement thereof.
For example, a detection probe oligomer may include a target-hybridizing sequence
that is from about 13 to about 40 nucleotides in length and is (i) contained in the
sequence of SEQ ID NO:196 or its complement and (ii) includes a sequence selected
from SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, and SEQ ID NO:178, including complements
thereof. In more specific variations, a detection probe oligomer target-hybridizing
sequence is (i) contained in the sequence of SEQ ID NO:197 or its complement and (ii)
includes the sequence of SEQ ID NO:174 or SEQ ID NO:175, including complements thereof.
In other variations, a detection probe oligomer target-hybridizing sequence is (i)
contained in the sequence of SEQ ID NO:196 or its complement and (ii) includes a sequence
selected from SEQ ID NO:177 and SEQ ID NO:178, including complements thereof. Particularly
suitable detection probe oligomer target-hybridizing sequences include SEQ ID NOs:131,
132, 135, 140, 145, 147-157, and 159-161, including complements thereof. Suitable
detection probes further include DNA equivalents and DNA/RNA chimerics of any of the
above
[0112] In some embodiments where a
Plasmodium sp. target region corresponds to a region of SEQ ID NO:180 from about nucleotide
position 1153, about nucleotide position 1169, or about nucleotide position 1182 to
about nucleotide position 1327, about nucleotide position 1354, or about nucleotide
position 1382, the oligomer combination includes a detection probe oligomer that specifically
hybridizes to a target region corresponding to a region of SEQ ID NO:180 from about
nucleotide position 1210 to about nucleotide position 1233 or the full complement
thereof. For example, a detection probe oligomer may include a target-hybridizing
sequence that is at least about 13 nucleotides in length and is (i) contained in the
sequence of SEQ ID NO:189 or its complement and (ii) includes a sequence selected
from SEQ ID NO:190 and SEQ ID NO:191, including complements thereof. Particularly
suitable detection probe oligomer target-hybridizing sequences include SEQ ID NOs:125-130
and 143, including complements thereof. Suitable detection probes further include
DNA equivalents and DNA/RNA chimerics of any of the above.
[0113] Table 3 shows exemplary combinations of detection probe target hybridizing sequences
together with first and second amplification oligomer target-hybridizing sequences
("Amp 1" and "Amp 2") for detection of
Plasmodium species target nucleic acid.
Table 3. Exemplary Combinations of Amplification Oligomer and Detection Probe Target-hybridizing
Sequences.
Amplification Oligomer THSs |
Detection Probe THS (SEQ ID NO) |
Amp 1 (SEQ ID NO) |
Amp 2 (SEQ ID NO) |
30 |
82 |
151 |
30 |
82 |
157 |
33 |
82 |
155 |
49 |
82 |
150 |
49 |
82 |
155 |
21 |
89 |
148 |
21 |
89 |
152 |
30 |
89 |
148 |
30 |
89 |
152 |
33 |
89 |
158 |
49 |
89 |
150 |
21 |
92 |
148 |
21 |
92 |
152 |
30 |
92 |
148 |
30 |
92 |
152 |
21 |
94 |
148 |
21 |
94 |
152 |
34 |
94 |
148 |
34 |
94 |
152 |
34 |
94 |
157 |
53 |
94 |
148 |
53 |
94 |
152 |
53 |
94 |
157 |
21 |
95 |
148 |
21 |
95 |
152 |
34 |
95 |
148 |
34 |
95 |
152 |
34 |
95 |
157 |
53 |
95 |
148 |
53 |
95 |
152 |
53 |
95 |
157 |
183 |
182 |
126 |
183 |
182 |
127 |
183 |
182 |
128 |
183 |
182 |
143 |
183 |
182 |
129 |
184 |
182 |
126 |
[0114] In some variations, an oligomer combination includes at least two detection probe
oligomers
(e.g., at least two specific detection probes as described herein). For example, where a
Plasmodium sp. target region corresponds to a region of SEQ ID NO:180 from about nucleotide
position 844 or about nucleotide position 910 to about nucleotide position 1038, about
nucleotide position 1051, about nucleotide position 1060, or about nucleotide position
1077, an oligomer combination may include (A) a first detection probe oligomer comprising
a target-hybridizing sequence that is (i) contained in the sequence of SEQ ID NO:197
or its complement and (ii) includes the sequence of SEQ ID NO: 175 or its complement,
and (B) a second detection probe oligomer comprising a target-hybridizing sequence
that is (i) contained in the sequence of SEQ ID NO:197 or its complement and (ii)
includes the sequence of SEQ ID NO:176 or its complement (including DNA equivalents
or DNA/RNA chimerics of the foregoing). Particularly suitable combinations of first
and second detection probe oligomers include a first detection probe oligomer comprising
the target-hybridizing sequence of SEQ ID NO: 157 or its complement, or a DNA equivalent
or DNA/RNA chimeric thereof, and a second detection probe oligomer comprising a target-hybridizing
sequence selected from SEQ ID NO: 148 and SEQ ID NO: 152, including complements, DNA
equivalents, and DNA/RNA chimerics thereof.
[0115] A detection probe oligomer may contain a 2'-methoxy backbone at one or more linkages
in the nucleic acid backbone. In some embodiments, the at least one detection probe
oligomer is provided in an amplicon detection reaction mixture.
[0116] Typically, a detection probe oligomer in accordance with the present invention further
includes a label. Particularly suitable labels include compounds that emit a detectable
light signal,
e.g., fluorophores or luminescent
(e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. More than
one label, and more than one type of label, may be present on a particular probe,
or detection may rely on using a mixture of probes in which each probe is labeled
with a compound that produces a detectable signal
(see, e.g., US Pat. Nos. 6,180,340 and
6,350,579,). Labels may be attached to a probe by various means including covalent linkages,
chelation, and ionic interactions, but preferably the label is covalently attached.
For example, in some embodiments, a detection probe has an attached chemiluminescent
label such as,
e.g., an acridinium ester (AE) compound
(see, e.g., US Patent Nos. 5,185,439;
5,639,604;
5,585,481; and
5,656,744;), which in typical variations is attached to the probe by a non-nucleotide linker
(see, e.g., US Patent Nos. 5,585,481;
5,656,744; and
5,639,604, particularly at column 10, line 6 to column 11, line 3, and Example 8;). In other
embodiments, a detection probe comprises both a fluorescent label and a quencher,
a combination that is particularly useful in fluorescence resonance energy transfer
(FRET) assays. Specific variations of such detection probes include,
e.g., a TaqMan detection probe (Roche Molecular Diagnostics) and a "molecular beacon"
(see, e.g., Tyagi et al., Nature Biotechnol. 16:49-53, 1998;
US Patent Nos. 5,118,801 and
5,312,728;).
[0117] A detection probe oligomer in accordance with the present invention may further include
a non-target-hybridizing sequence. Specific embodiments of such detection probes include,
for example, probes that form conformations held by intramolecular hybridization,
such as conformations generally referred to as hairpins. Particularly suitable hairpin
probes include a "molecular torch"
(see, e.g., US Patent Nos. 6,849,412;
6,835,542;
6,534,274; and
6,361,945,) and a "molecular beacon"
(see, e.g., Tyagi
et al., supra; US 5,118,801 and
US 5,312,728,
supra). Methods for using such hairpin probes are well-known in the art.
[0118] In yet other embodiments, a detection probe is a linear oligomer that does not substantially
form conformations held by intramolecular bonds. In specific variations, a linear
detection probe oligomer includes a chemiluminescent compound as the label, preferably
an acridinium ester (AE) compound.
[0119] Also provided by the present invention are detection probe oligomers, capture probe
oligomers, and combinations thereof as described herein.
[0120] In other aspects, the present invention provides methods for detecting the presence
or absence of
Plasmodium sp. in a sample from a subject. Such methods generally include performing a nucleic-acid-based
detection assay for the specific detection in the sample of
Plasmodium sp. nucleic acid. A nucleic-acid-based detection assay for specific detection of
Plasmodium sp. may use any one or more
Plasmodium sp.-specific oligomers as described herein
(e.g., an oligomer combination as described herein comprising at least two amplification
oligomers; or a detection probe or combination of detection probes as described herein).
A positive signal from a nucleic-acid-based detection assay in accordance with the
present invention is indicative of the presence of one or more of
Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and/or
Plasmodium vivax in a sample.
[0121] In some embodiments of a method comprising the use of a nucleic-acid-based detection
assay, an amplification-based assay is used to detect
Plasmodium sp. Such amplification-based assay methods generally include performing a nucleic
acid amplification of an
Plasmodium sp. target region and detecting the amplified product
(e.g., by specifically hybridizing the amplified product with a nucleic acid detection probe
that provides a signal to indicate the presence of
Plasmodium sp. in the sample). The amplification step includes contacting the sample with one
or more amplification oligomers specific for a target sequence in a
Plasmodium sp. target nucleic acid to produce an amplified product if
Plasmodium sp. nucleic acid is present in the sample. In particular embodiments, a combination
of at least two amplification oligomers as described herein are used at the amplification
step. Amplification synthesizes additional copies of the target sequence or its complement
by using at least one nucleic acid polymerase and an amplification oligomer to produce
the copies from a template strand (
e.g., by extending the sequence from a primer using the template strand). Suitable amplification
methods include, for example, replicase-mediated amplification, polymerase chain reaction
(PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), and transcription-mediated
or transcription-associated amplification (TMA). Such amplification methods are well-known
in the art
(see, e.g., discussion of amplification methods in Definitions section,
supra) and are readily used in accordance with the methods of the present disclosure.
[0122] Detection of the amplified products may be accomplished by a variety of methods to
detect a signal specifically associated with the amplified target sequence. The nucleic
acids may be associated with a surface that results in a physical change, such as
a detectable electrical change. Amplified nucleic acids may be detected by concentrating
them in or on a matrix and detecting the nucleic acids or dyes associated with them
(e.g., an intercalating agent such as ethidium bromide or cyber green), or detecting an
increase in dye associated with nucleic acid in solution phase. Other methods of detection
may use uses a hybridizing step that includes contacting the amplified product with
at least one detection probe configured to specifically hybridize to a sequence in
the amplified product and detecting the presence of the probe: product complex, or
by using a complex of probes that may amplify the detectable signal associated with
the amplified products (
e.g.,
US Patent Nos. 5,424,413;
5,451,503; and
5,849,481). Directly or indirectly labeled probes that specifically associate with the amplified
product provide a detectable signal that indicates the presence of the target nucleic
acid in the sample. In some embodiments, a method utilizing an amplification-based
assay for detection of
Plasmodium species utilizes one or more detection probe oligomers as described herein for detection
of an amplified product.
[0123] Detection probes that hybridize to the complementary amplified sequences may be DNA
or RNA oligomers, or oligomers that contain a combination of DNA and RNA nucleotides,
or oligomers synthesized with a modified backbone, e.g.
, an oligomer that includes one or more 2'-methoxy substituted ribonucleotides. Probes
used for detection of the amplified sequences may be unlabeled and detected indirectly
(
e.g., by binding of another binding partner to a moiety on the probe) or may be labeled
with a variety of detectable labels. In some embodiments of a method for detecting
Plasmodium species, such as in certain embodiments using transcription-mediated amplification
(TMA), the detection probe is a linear chemiluminescently labeled probe such as,
e.g., a linear acridinium ester (AE) labeled probe. The detection step may also provide
additional information on the amplified sequence, such as,
e.g., all or a portion of its nucleic acid base sequence. Detection may be performed after
the amplification reaction is completed, or may be performed simultaneously with amplifying
the target region,
e.g., in real time. In one embodiment, the detection step allows homogeneous detection,
e.g., detection of the hybridized probe without removal of unhybridized probe from the
mixture
(see, e.g., US Patent Nos. 5,639,604 and
5,283,174).
[0124] In embodiments that detect the amplified product near or at the end of the amplification
step, a linear detection probe may be used to provide a signal to indicate hybridization
of the probe to the amplified product. One example of such detection uses a luminescentally
labeled probe that hybridizes to target nucleic acid. Luminescent label is then hydrolyzed
from non -hybridized probe. Detection is performed by chemiluminescence using a luminometer
(see, e.g., International Patent Application Pub. No.
WO 89/002476). In other embodiments that use real-time detection, the detection probe may be a
hairpin probe such as, for example, a molecular beacon, molecular torch, or hybridization
switch probe that is labeled with a reporter moiety that is detected when the probe
binds to amplified product. Such probes may comprise target-hybridizing sequences
and non-target-hybridizing sequences. Various forms of such probes have been described
previously
(see, e.g., US Patent Nos. 5,118,801 ;
5,312,728;
5,925,517;
6,150,097;
6,849,412;
6,835,542;
6,534,274; and
6,361,945; and
US Patent Application Pub. Nos. 20060068417A1 and
20060194240A1).
[0125] Some amplification methods that use TMA amplification include the following steps.
Briefly, the target nucleic acid that contains the sequence to be amplified is provided
as single stranded nucleic acid
(e.g., ssRNA or ssDNA). Those skilled in the art will appreciate that conventional melting
of double stranded nucleic acid (
e.g., dsDNA) may be used to provide single-stranded target nucleic acids. A promoter
primer binds specifically to the target nucleic acid at its target sequence and a
reverse transcriptase (RT) extends the 3' end of the promoter primer using the target
strand as a template to create a cDNA copy of the target sequence strand, resulting
in an RNA:DNA duplex. An RNase digests the RNA strand of the RNA:DNA duplex and a
second primer binds specifically to its target sequence, which is located on the cDNA
strand downstream from the promoter primer end. RT synthesizes a new DNA strand by
extending the 3' end of the second primer using the first cDNA template to create
a dsDNA that contains a functional promoter sequence. An RNA polymerase specific for
the promoter sequence then initiates transcription to produce RNA transcripts that
are about 100 to 1000 amplified copies ("amplicons") of the initial target strand
in the reaction. Amplification continues when the second primer binds specifically
to its target sequence in each of the amplicons and RT creates a DNA copy from the
amplicon RNA template to produce an RNA:DNA duplex. RNase in the reaction mixture
digests the amplicon RNA from the RNA:DNA duplex and the promoter primer binds specifically
to its complementary sequence in the newly synthesized DNA. RT extends the 3' end
of the promoter primer to create a dsDNA that contains a functional promoter to which
the RNA polymerase binds to transcribe additional amplicons that are complementary
to the target strand. The autocatalytic cycles of making more amplicon copies repeat
during the course of the reaction resulting in about a billion-fold amplification
of the target nucleic acid present in the sample. The amplified products may be detected
in real-time during amplification, or at the end of the amplification reaction by
using a probe that binds specifically to a target sequence contained in the amplified
products. Detection of a signal resulting from the bound probes indicates the presence
of the target nucleic acid in the sample.
[0126] In some embodiments, the method utilizes a "reverse" TMA reaction. In such variations,
the initial or "forward" amplification oligomer is a priming oligonucleotide that
hybridizes to the target nucleic acid in the vicinity of the 3'-end of the target
region. A reverse transcriptase (RT) synthesizes a cDNA strand by extending the 3'-end
of the primer using the target nucleic acid as a template. The second or "reverse"
amplification oligomer is a promoter primer or promoter provider having a target-hybridizing
sequence configured to hybridize to a target-sequence contained within the synthesized
cDNA strand. Where the second amplification oligomer is a promoter primer, RT extends
the 3' end of the promoter primer using the cDNA strand as a template to create a
second, cDNA copy of the target sequence strand, thereby creating a dsDNA that contains
a functional promoter sequence. Amplification then continues essentially as described
above for initiation of transcription from the promoter sequence utilizing an RNA
polymerase. Alternatively, where the second amplification oligomer is a promoter provider,
a terminating oligonucleotide, which hybridizes to a target sequence that is in the
vicinity to the 5'-end of the target region, is typically utilized to terminate extension
of the priming oligomer at the 3'-end of the terminating oligonucleotide, thereby
providing a defined 3'-end for the initial cDNA strand synthesized by extension from
the priming oligomer. The target-hybridizing sequence of the promoter provider then
hybridizes to the defined 3'-end of the initial cDNA strand, and the 3'-end of the
cDNA strand is extended to add sequence complementary to the promoter sequence of
the promoter provider, resulting in the formation of a double-stranded promoter sequence.
The initial cDNA strand is then used a template to transcribe multiple RNA transcripts
complementary to the initial cDNA strand, not including the promoter portion, using
an RNA polymerase that recognizes the double-stranded promoter and initiates transcription
therefrom. Each of these RNA transcripts is then available to serve as a template
for further amplification from the first priming amplification oligomer.
[0127] In some embodiments of a method comprising the use of a nucleic-acid-based detection
assay, a non-amplification-based assay is used to detect
Plasmodium sp. In some such embodiments, the non-amplification-based assay is a hybridization
assay comprising the hybridization of a specific detection probe to a target nucleic
acid. Methods for conducting polynucleotide hybridization assays have been well developed
in the art. Hybridization assay procedures and conditions will vary depending on the
application and are selected in accordance with the general binding methods known,
including those referred to in,
e.g., Maniatis et al, Molecular Cloning: A Laboratory Manual (3rd ed. Cold Spring Harbor,
N.Y., 2002), and
Berger and Kimmel, Methods in Enzymology, Vol. 152 Guide to Molecular Cloning Techniques
(Academic Press, Inc., San Diego, Calif., 1987, ). Generally, the probe and sample are mixed under conditions that will permit specific
nucleic acid hybridization, and specific hybridization of the probe to its respective
target is then detected. Nucleic acid hybridization is adaptable to a variety of assay
formats. One suitable format is the sandwich assay format, which is particularly adaptable
to hybridization under non-denaturing conditions. A primary component of a sandwich-type
assay is a solid support, which has adsorbed to it or covalently coupled to it immobilized
nucleic acid probe that is unlabeled and complementary to one portion of the DNA sequence.
Target nucleic acid is hybridized to the immobilized probe, and a second, labeled
detection probe - which is complementary to a second and different region of the same
DNA strand to which the immobilized, unlabeled nucleic acid probe is hybridized -
is hybridized to the [target nucleic acid]: [immobilized probe] duplex to detect the
target nucleic acid. Another exemplary format utilizes electrochemical detection of
target nucleic acids hybridized to unlabeled detection probes immobilized on a suitable
electrode surface as a signal transducer.
See, e.g., Drummond et al., Nat. Biotechnol. 21: 1192, 2003;
Gooding, Electroanalysis 14: 1149, 2002;
Wang, Anal. Chim. Acta 469:63, 2002;
Cagnin et al., Sensors 9:3122, 2009;
Katz and Willner, Electroanalysis 15:913, 2003;
Daniels and Pourmand, Electroanalysis 19: 1239, 2007.
[0128] In certain embodiments of a method for detecting
Plasmodium species comprising a hybridization assay, the hybridization assay utilizes one or
more detection probe oligomers as described herein.
[0130] Appropriate conditions for flap endonuclease reactions are either known or can be
readily determined using methods known in the art
(see, e.g., Kaiser et al., J. Biol. Chem. 274:2138-721394, 1999). Exemplary flap endonucleases that may be used in the method include
Thermus aquaticus DNA polymerase I,
Thermus thermophilus DNA polymerase I, mammalian FEN-1,
Archaeoglobus fulgidus FEN-1,
Methanococcus jannaschii FEN-1,
Pyrococcus fiiriosus FEN-1,
Methanobacterium thermoautotrophicum FEN-1,
Thermus thermophilus FEN-1, CLEAVASE
® (Hologic, Inc., Madison, WI),
S.
cerevisiae RTH1, S.
cerevisiae RAD27,
Schizosaccharomyces pombe rad2, bacteriophage T5 5' -3' exonuclease,
Pyrococcus horikoshii FEN-1, human endonuclease 1, calf thymus 5'-3' exonuclease, including homologs thereof
in eubacteria, eukaryotes, and archaea, such as members of the class II family of
structure-specific enzymes, as well as enzymatically active mutants or variants thereof.
Descriptions of flap endonucleases can be found in, for example,
Lyamichev et al., Science 260:778-783, 1993;
Eis et al., Nat. Biotechnol. 19:673-676, 2001;
Shen et al., Trends in Bio. Sci. 23: 171 -173, 1998;
Kaiser et al., J. Biol. Chem. 274:21387-21394, 1999;
Ma etal., J. Biol. Chem. 275:24693-24700, 2000;
Allawi et al., J. Mol. Biol. 328:537-554, 2003;
Sharma et al., J. Biol. Chem. 278:23487-23496, 2003; and
Feng et al., Nat. Struct. Mol. Biol. 11 :450-456, 2004.
[0131] In certain variations, a cleavage-based assay detects an RNA target nucleic acid
of
Plasmodium sp., and the cleavage-based assay utilizes a flap endonuclease that is capable of
cleaving and RNA:DNA linear duplex structure. In some alternative embodiments, a cleavage-based
assay detects a DNA target nucleic acid
of Plasmodium sp., and the cleavage-based assay utilizes a flap endonuclease that is capable of
cleaving and DNA:DNA linear duplex structure. Exemplary flap endonucleases capable
of cleaving RNA:DNA duplexes include polymerasedeficient 5' nucleases of the genus
Thermus as well as certain CLEAVASE
® enzymes (Hologic, Inc., Madison, WI) such as, for example, CLEAVASE
® BN (BstX-Notl deletion of Taq polymerase, see
US Patent No. 5,614,402), CLEAVASE
® II ("AG" mutant of full length Taq polymerase, see
US Patent No. 5,614, 402), CLEAVASE
® VII (synthesis-deficient mutation of full length
Thermus thermophilus polymerase), CLEAVASE
® IX (polymerase deficient mutant of the Tth DNA polymerase), and CLEAVASE
® XII (polymerase deficient chimeric polymerase constructed from fragments of taq DNA
polymerase and Tth DNA polymerase). Exemplary flap endonucleases capable of cleaving
DNA:DNA duplexes include the flap endonucleases indicated above, as well as CLEAVASE
® 2.0 (
Archaeoglobus fulgidus FEN-1), CLEAVASE
® 2.1 (
Archaeoglobus fulgidus FEN-1 with 6 histidines on the C-terminus), CLEAVASE
® 3.0 (
Archaeoglobus veneficus FEN-1), and CLEAVASE
® 3.1 (
Archaeoglobus veneficus FEN-1 with 6 histidines on the C-terminus).
[0132] In some embodiments, a cleavage-based assay detects an RNA target nucleic acid of
Plasmodium sp., and the assay includes a step for synthesizing a DNA complement of an RNA target
region, which cDNA strand is then hybridized to overlapping first and second probe
oligonucleotides to form a linear duplex cleavage structure for cleavage by the flap
endonuclease. Reaction conditions for synthesizing cDNA from an RNA template, using
an RNA-dependent DNA polymerase (reverse transcriptase), are well-known in the art.
[0133] In certain embodiments utilizing a nucleic-acid-based detection assay, the method
further includes purifying the
Plasmodium sp. target nucleic acid from other components in the sample. Such purification may
include methods of separating and/or concentrating organisms contained in a sample
from other sample components. In particular embodiments, purifying the target nucleic
acid includes capturing the target nucleic acid to specifically or non-specifically
separate the target nucleic acid from other sample components. Non-specific target
capture methods may involve selective precipitation of nucleic acids from a substantially
aqueous mixture, adherence of nucleic acids to a support that is washed to remove
other sample components, or other means of physically separating nucleic acids from
a mixture that contains
Plasmodium sp. nucleic acid and other sample components. In some embodiments, purification includes
lysing a sample of cells such as, for example, blood cells
(e.g., red blood cells) and purifying any
Plasmodium sp. target nucleic acid from the lysed cell sample. Exemplary lysis reagents and
methods for used in accordance with the present invention are described in
US Pat. No. 10,093,989 and
PCT Pub. No. WO 2017/189746.
[0134] In some embodiments, a target nucleic acid of
Plasmodium sp. is separated from other sample components by hybridizing the target nucleic acid
to a capture probe oligomer. The capture probe oligomer comprises a target-hybridizing
sequence configured to specifically or non-specifically hybridize to a target nucleic
acid so as to form a [target nucleic acid]: [capture probe] complex that is separated
from other sample components. Capture probes comprising target-hybridizing sequences
suitable for non-specific capture of target nucleic acids are described in,
e.g., PCT Pub. No. WO 2008/016988. In some specific variations comprising target-hybridizing sequence(s) configured
to specifically hybridize to a
Plasmodium sp. target nucleic acid, a
Plasmodium sp.-specific capture probe comprises a target-hybridizing sequence that is up to
about 30 contiguous nucleotides in length and includes a sequence substantially corresponding
to a sequence selected from SEQ ID NOs:11-15, 17, 19, and 20
(e.g., a target-hybridizing sequence comprising or consisting of a sequence selected from
SEQ ID NOs:11-15, 17, 19, and 20, including DNA equivalents and DNA/RNA chimerics
thereof). In a preferred variation, the capture probe binds the [target nucleic acid]:[capture
probe] complex to an immobilized probe to form a [target nucleic acid]:[capture probe]:
[immobilized probe] complex that is separated from the sample and, optionally, washed
to remove non-target sample components
(see, e.g., US Patent Nos. 6,110,678;
6,280,952; and
6,534,273). In such variations, the capture probe oligomer further comprises a sequence or
moiety that binds the capture probe, with its bound target sequence, to an immobilized
probe attached to a solid support, thereby permitting the hybridized target nucleic
acid to be separated from other sample components.
[0135] In more specific embodiments, the capture probe oligomer includes a tail portion
(e.g., a 3' tail) that is not complementary to target nucleic acid but that specifically
hybridizes to a sequence on the immobilized probe, thereby serving as the moiety allowing
the target nucleic acid to be separated from other sample components, such as previously
described in,
e.g., U.S. Patent No. 6,110,678. Any sequence may be used in a tail region, which is generally about 5 to 50 nt long,
and preferred embodiments include a substantially homopolymeric tail of about 10 to
40 nt (
e.g., A10 to A40), more preferably about 14 to 33 nt (
e.g., A14 to A30 or T3A14 to T3A30), that bind to a complementary immobilized sequence
(
e.g., poly-T) attached to a solid support,
e.g., a matrix or particle. In some such embodiments comprising target-hybridizing sequence(s)
configured to specifically hybridize to
Plasmodium sp. target nucleic acid, a
Plasmodium sp.-specific capture probe comprises or consists of a nucleotide sequence selected
from SEQ ID NOs:1-5, 7, 9, and 10.
[0136] Target capture typically occurs in a solution phase mixture that contains one or
more capture probe oligomers that hybridize to the target nucleic acid under hybridizing
conditions, usually at a temperature higher than the Tm of the [tail sequence]:[immobilized
probe sequence] duplex. For embodiments comprising a capture probe tail, the [target
nucleic acid]:[capture probe] complex is captured by adjusting the hybridization conditions
so that the capture probe tail hybridizes to the immobilized probe, and the entire
complex on the solid support is then separated from other sample components. The support
with the attached [immobilized probe]: [capture probe]: [target nucleic acid] may
be washed one or more times to further remove other sample components. Preferred embodiments
use a particulate solid support, such as paramagnetic beads, so that particles with
the attached [target nucleic acid]:[capture probe]:[immobilized probe] complex may
be suspended in a washing solution and retrieved from the washing solution, preferably
by using magnetic attraction. In embodiments of the method comprising the use of an
amplification-based detection assay, to limit the number of handling steps, a target
nucleic acid may be amplified by simply mixing the target nucleic acid in the complex
on the support with amplification oligomers and proceeding with amplification steps.
[0137] In accordance with the present disclosure, detecting the presence or absence of
Plasmodium sp. may be performed separately
(e.g., in a separate reaction vessel), or performed together with another assay as a multiplex
reaction system. Accordingly, in some embodiments, a method as described herein utilizes
a multiplex reaction, where the reaction mix contains reagents for assaying multiple
(
e.g., at least two, three, four, or more) different target sequences in parallel. In these
cases, a reaction mix may contain multiple different target-specific oligonucleotides
for performing the detection assay. For example, in a method utilizing an amplification-based
detection assay, a multiplex reaction may contain multiple sets (
e.g., multiple pairs) of amplification oligomers (for example, multiple pairs of PCR primers
or multiple pairs of TMA amplification oligomers (
e.g., for TMA, multiple pairs of promoter primer and non-promoter primer, or multiple
pairs of promoter provider and non-promoter primer)). In other embodiments utilizing
a cleavage-based detection assay, a multiplex reaction may contain multiple probe
oligonucleotides having different flaps, multiple different overlapping probe oligonucleotides,
and multiple different FRET cassettes for detecting the different flaps, once they
are cleaved.
[0138] The oligomer combination described herein may be in the form of a reaction mixture
or a kit comprising the oligomers. The reaction mixture or kit may further include
a number of optional components such as, for example, capture probe nucleic acids
or arrays of capture probe nucleic acids. For an amplification reaction mixture, the
reaction mixture will typically include other reagents suitable for performing
in vitro amplification such as,
e.g., buffers, salt solutions, appropriate nucleotide triphosphates (
e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP), and/or enzymes (
e.g., reverse transcriptase, and/or RNA polymerase), and will typically include test
sample components, in which a
Plasmodium sp. target nucleic acid may or may not be present. A kit comprising an oligomer combination
for amplification of
Plasmodium sp. may also include other reagents suitable for performing
in vitro amplification such as,
e.g., buffers, salt solutions, appropriate nucleotide triphosphates
(e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP), and/or enzymes
(e.g., reverse transcriptase, and/or RNA polymerase). For an oligomer combination (
e.g., reaction mixture or kit) that includes a detection probe together with an amplification
oligomer combination targeting a common target nucleic acid, selection of amplification
oligomers and detection probe oligomers are linked by a common target region (
i.e., the combination will include a probe that binds to a sequence amplifiable by the
amplification oligomer combination).
[0139] The compositions, methods, reaction mixtures, systems, kits and the like for detection
of
Plasmodium nucleic acids are further illustrated by the following non-limiting examples.
EXAMPLES
[0140] "Parasite Transport Solution" generally refers to a solution formulated to preserve
a sample, and in some instances formulated to at least partially lyse one or more
cell types in a sample. One exemplary parasite transport solution comprises 15 mM
sodium phosphate monobasic, 15 mM sodium phosphate dibasic, 1 mM EDTA, 1 mM EGTA,
and 110 mM lithium lauryl sulfate (LLS), at pH 6.7. Another exemplary parasite transport
solution comprises an aqueous solution of 100 mM TRIS, 30 mM magnesium chloride, and
6% (v/v) LLS, at pH 7.5. A further exemplary parasite transport solution comprises
an aqueous solution of 14 mM sodium bicarbonate, 250 mM ammonium chloride, 5% (v/v)
LLS, and 0.1 mM EDTA, at a pH of 7.4. Other formulations of parasite transport solutions
may function equally well.
[0141] "Target Capture Reagent" generally refers to a solution containing a number of components
that facilitate capture of a nucleic acid from a solution. One exemplary Target Capture
Reagent comprises 250 mM HEPES, 310 mM lithium hydroxide, 1.88 M lithium chloride,
100 mM EDTA, at pH 6.4, and 250 µg/ml of magnetic particles (1 micron SERA-MAG^TM
MG-CM particles, GE Healthcare Lifesciences) with dT
14 oligomers covalently bound thereto. Another exemplary Target Capture Reagent comprises
790 mM HEPES, 453 mM lithium hydroxide, 10% w/v LLS, 230 mM Succinic Acid, 0.03% w/v
Foam Ban MS-575, and 0.0125% w/v of magnetic particles (1 micron SERA-MAC^TM MG-CM
particles, GE Healthcare Lifesciences) with dT
14 oligomers covalently bound. Other formulations of Target Capture Reagent may function
equally as well.
[0142] "Wash Solution" generally refers to a solution containing 10 mM HEPES, 150 mM sodium
chloride, 6.5 mM sodium hydroxide, 1 mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v) methyl
paraben, 0.01% (w/v) propyl paraben, and 0.1% (w/v) sodium lauryl sulfate, at pH 7.5.
[0143] "Probe Reagent" generally refers to a solution containing one or more labeled detection
probes. One exemplary Probe Reagent is a solution made up of from about 75 to about
100 mM lithium succinate, 2% (w/v) LLS, 15 mM mercaptoethanesulfonate, 1.2 M lithium
chloride, 20 mM EDTA, and 3% (v/v) ethanol, at pH 4.7. Another exemplary Probe Reagent
is a solution made up of from about 75 to about 100 mM succinic acid, 3.5% (w/v) LLS,
75 mM lithium hydroxide, 15 mM aldrithiol-2, 1.0 M lithium chloride, 1 mM EDTA, and
3.0% (v/v) ethanol, at pH 4.1-4.3. Other formulations may perform equally as well.
[0144] "Amplification Reagent" generally refers to a concentrated mixture of reaction components
to facilitate amplification reactions. An Amplification Reagent will comprise a number
of different reagents at various concentrations depending on factors such as for example
amplification type (PCR, TMA, etc.), target nucleic acids (GC content), and the like.
One exemplary Amplification Reagent comprises 47.6 mM Na-HEPES, 12.5 mM N-acetyl-L-cysteine,
2.5% TRITON
™ X-100, 54.8 mM KCl, 23 mM MgCl2, 3 mM NaOH, 0.35 mM of each dNTP (dATP, dCTP, dGTP,
dTTP), 7.06 mM rATP, 1.35 mM rCTP, 1.35 mM UTP, 8.85 mM rGTP, 0.26 mM Na2EDTA, 5%
v/v glycerol, 2.9% trehalose, 0.225% ethanol, 0.075% methylparaben, 0.015% propylparaben,
and 0.002% Phenol Red, at pH 7.5-7.6. Another exemplary Amplification Reagent comprises
19.1 mM Trizma Base, 7.5mM Trizma Hydrochloride, 23.3 mM KCl, 21.5 mM MgCl2, 1 mM
of each dNTP (dATP, dCTP, dGTP, dTTP), 6.5 mM rATP, 4.0 mM rCTP, 4.0 mM UTP, 6.5 mM
rGTP, 3.33% v/v glycerol, 0.05 mM Zinc Acetate, 6 ppm Pro Clin 300 preservative, at
pH 8.25-8.45. Other formulations of amplification reagent may function equally well.
Primers may be added to the amplification reagent or added to amplification reactions
separate from the amplification reagent. Enzymes in an amplification reagent can include
one or more of Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) and bacteriophage
T7 RNA polymerase for which units are functionally defined as: 1 U of MMLV-RT incorporates
1 nmol of dTTP in 10 min at 37C using 200-400 micromolar oligo dT-primed poly(A) as
template, and 1 U of T7 RNA polymerase incorporates 1 nmol of ATP into RNA in 1 hr
at 37C using a DNA template containing a T7 promoter.
[0145] "Hybridization Reagent" generally refers to a solution made up of reagents having
concentrations in the range of about: 75-100 mM succinic acid, 2%-3.5% (w/v) LLS,
75-100 mM lithium hydroxide, 14-16 mM aldrithiol-2, 1.0-1.2 M lithium chloride, 20-1000
mM EDTA, and 2.0-4.0% (v/v) ethanol, at pH 4-5 Other formulations for a Hybridization
Reagent may function equally well.
[0146] "Selection Reagent" generally refers to a solution containing 600 mM boric acid,
182.5 mM sodium hydroxide, 1% (v/v) octoxynol (TRITON
® X-100), at pH 8.5.
[0147] "Detection Reagents" include "Detect Reagent I," which generally refers to a solution
containing 1 mM nitric acid and 32 mM hydrogen peroxide, and "Detect Reagent II,"
which generally refers to a solution of 1.5 M sodium hydroxide.
Example 1
[0148] Primer screening was performed using Transcription-Mediated Amplification (TMA) on
the manual Procleix Enhanced Semi-automated System (eSAS) using
Plasmodium falciparum in vitro transcript (IVT) . An assay rack consisted of 10 rows of Ten-tube units (TTUs). Seventy
five microliters (75 µL) of Amplification Reagent and 5 picomoles of each T7 promoter
provider oligonucleotide and non-T7 primer oligonucleotide were added to the appropriate
tubes on the rack such that each combination of amplification oligomers were tested
with three replicates of
P. falciparum IVT at 30 and 10 copies per reaction and two replicates of
B. microti IVT at 1,000,000 copies per reaction, where applicable.
B. microti was included in initial screening as a cross reactivity specimen due to the conserved
regions between
Babesia and
Plasmodium. It is necessary to determine that amplification and detection systems are specific
to
Plasmodium. To achieve the target copies per reaction, 10 µL of
P. falciparum IVT at 3 c/µL or 1 c/µL diluted in a buffer was spiked into the appropriate tubes,
and 10 µL of
B.
microti IVT at 100,000 c/µL diluted in a buffer were spiked into the appropriate tubes. Various
combinations of primers were tested. This set-up allows for 10 primer combinations
to be tested per rack. Once the primer combinations and IVTs were spiked, 200 µL of
oil was added to each tube and then the rack was covered with sealing cards and vortexed
for a minimum of 20 seconds.
[0149] The rack was then incubated in a water bath at 60±1°C for 10±1 minutes followed by
incubation in a 41.5±1°C water bath between 9 and 20 minutes. While the rack remained
in the water bath, the sealing cards were removed and 25µL of commercially available
Procleix Ultrio Plus enzyme reagent (Grifols Diagnostic Solutions Inc.) was added
to each reaction tube and then covered again with sealing cards. The rack was gently
shaken to mix and then covered again with sealing cards and incubated for another
60±5 minutes in the 41.5±1°C water bath.
[0150] After incubation completed, the rack was transferred to the hybridization protection
assay (HPA) area where the sealing cards were removed. 100µL of Probe reagent consisting
of an Acridinium-Ester (AE) labeled probe added at a total desired concentration of
at least 2.5e6 Relative Light Units (RLU) per reaction to a Hybridization reagent.
Probe reagent was then added to the appropriate reaction tubes. The tubes were covered
with sealing cards and the rack was vortexed for a minimum of 20 seconds after which
the rack was incubated in a water bath at 61±2°C for 15±1 minutes.
[0151] The rack was removed from the water bath, the sealing cards removed, and 250 µL of
commercially available Procleix Ultrio Plus Selection reagent (Grifols Diagnostic
Solutions Inc.) was added to each tube. The tubes were covered with sealing cards
and vortexed for a minimum of 20 seconds and then returned to the 61±2°C water bath
and incubated for 10±1 minutes. After incubation, the rack was allowed to cool in
a 23±4°C water bath for a minimum of 10 minutes.
[0152] For detection the TTUs are removed from the rack and loaded on to the automated Leader
instrument for subsequent light off using commercially available Procleix Auto Detect
1 and 2 reagents (Grifols Diagnostic Solutions Inc.) and the results were exported
for analysis of the signal in Relative Light Units (RLU).
[0153] AE-labeled probes screened in this example are shown in Table 4 below. Probes were
screened with amplification oligomer pairs in four experimental groups: Group 1, Groups
2a and 2b, and Group 3.
Table 4.
Probe # |
SEQ ID NO: |
2MeAE linker site |
1 |
159 |
7,8 |
3 |
159 |
9,10 |
4 |
151 |
6,7 |
5 |
151 |
7,8 |
6 |
151 |
8,9 |
7 |
150 |
8,9 |
8 |
155 |
7,8 |
12 |
160 |
13,14 |
14 |
147 |
11,12 |
15 |
157 |
10,11 |
16 |
156 |
10,11 |
20 |
158 |
6,7 |
21 |
158 |
7,8 |
22 |
158 |
10,11 |
[0154] Group 1. Probes 1, 3, 4-6, 12, and 14-16 were each tested with SEQ ID NO:59 (T7 promoter
provider oligomer) and SEQ ID NO:30 (non-T7 oligomer). The results of this probe screen
are shown in Table 5 below. Candidates showed no cross-reactivity with
B. microti IVT at 1e6 copies per reaction. In addition, probes having the same nucleotide sequence
but with different 2MeAE linker sites
(see, e.g., probes 1 and 3;
see also probes 4, 5, and 6) performed well irrespective of their different labelling.
Table 5.
|
P. fal 30 c/rxn |
P. fal 30 c/rxn |
P. fal 30 c/rxn |
P. fal 10 c/rxn |
P. fal 10 c/rxn |
P. fal 10 c/rxn |
B.mic 1e6 c/rxn |
B.mic 1e6 c/rxn |
B.mic 1e6c/c/rxn |
Probe 1 |
1,222,972 |
1,157,299 |
1,201,553 |
927,342 |
1,093,944 |
98,711 |
919 |
985 |
871 |
Probe 3 |
1,179,477 |
1,192,980 |
1,155,140 |
1,130,568 |
854,803 |
1,206,816 |
1,515 |
1,640 |
1,708 |
Probe 5 |
816,162 |
815,694 |
774,947 |
708,438 |
745,288 |
519,080 |
1,562 |
1,253 |
1,322 |
Probe 4 |
965,152 |
1,019,364 |
1,043,712 |
767,525 |
957,437 |
905,300 |
1,203 |
1,565 |
1,276 |
Probe 6 |
1,179,590 |
1,182,350 |
1,136,391 |
1,034,114 |
1,098,623 |
856,584 |
2,211 |
3,484 |
2,473 |
Probe 12 |
1,801,146 |
1,739,366 |
1,606,426 |
698,553 |
1,610,398 |
1,515,342 |
1,540 |
1,278 |
2,523 |
Probe 14 |
1,017,636 |
997,356 |
1,015,960 |
951,505 |
830,243 |
909,076 |
870 |
865 |
1,470 |
Probe 15 |
1,116,536 |
1,203,192 |
1,038,531 |
1,093,438 |
958,900 |
891,744 |
1,174 |
1,019 |
1,803 |
Probe 16 |
182,425 |
195,626 |
73,184 |
141,723 |
47,725 |
52,841 |
4,155 |
2,440 |
2,629 |
|
|
NEG |
Probe 1 |
Probe 3 |
Probe 4 |
1,108 |
1,046 |
1,310 |
1,935 |
1,749 |
1,784 |
1,319 |
1,489 |
1,388 |
Probe 4 |
Probe 6 |
Probe 12 |
2,614 |
1,194 |
2,321 |
2,673 |
2,935 |
4,364 |
1,473 |
1,474 |
2,528 |
Probe 14 |
Probe 15 |
Probe 16 |
1,3111 |
1,597, |
978 |
1,359, |
1,171 |
1,475 |
3,940 |
1,430 |
1,869 |
[0155] Groups 2a and 2b. In Group 2a, the following primer/probe combinations were tested: each of probes
8 and 20-22 paired with SEQ ID NO:59 and SEQ ID NO:33 (T7/NT7), each of probes 7 and
20-22 paired with SEQ ID NO:59 and SEQ ID NO:52 (T7/NT7), and each of probes 7 and
8 paired with SEQ ID NO:59 and SEQ ID NO:49 (T7/NT7). In Group 2b, the following primer/probe
combinations were tested: each of probes 20-22 paired with SEQ ID NO:66 and SEQ ID
NO:33 (T7/NT7), each of probes 7 and 20-22 paired with SEQ ID NO:66 and SEQ ID NO:52
(T7/NT7), and probe 7 paired with SEQ ID NO:66 and SEQ ID NO:49 (T7/NT7).
[0156] The results of this probe screen are shown in Tables 6 and 7 below. Systems with
the non-T7 oligomer of SEQ ID NO:52 did not amplify
Plasmodium. While systems with the T7/NT7 oligomer pair of SEQ ID NO:59/SEQ ID NO:33 paired with
any of Probes 20-22 did not detect
Plasmodium, these probes detect
Plasmodium when used with the T7/NT7 oligomer pair of SEQ ID NO:66/SEQ ID NO:33.
Table 6.
T7/NT7 + Probe |
P. fal 30 c/rxn |
P. fal 30 c/rxn |
P. fal 30 c/rxn |
P. fal 10 c/rxn |
P. fal 10 c/rxn |
P. fal 10 c/rxn |
B. mic 1e6 c/rxn |
B.mic 1e6 c/rxn |
NEG |
NEG |
59/49 +Probe 7 |
1,414,377 |
1,429,342 |
1,423,366 |
1,293,402 |
1,271,942 |
1,347,111 |
1,967 |
1,113 |
1,651 |
1,603 |
59/52 +Probe 7 |
15,998 |
92,089 |
4,310 |
676 |
1,465 |
3,151 |
1,473 |
3,105 |
793 |
1,315 |
59/49 +Probe 8 |
1,556,915 |
1,585,955 |
1,594,418 |
1,523,106 |
1,419,616 |
1,564,211 |
7,048 |
2,935 |
3,710 |
5,601 |
59/33 +Probe 8 |
1,026,164 |
1,188,939 |
1,511,173 |
1,022,388 |
1,030,495 |
421,293 |
2,804 |
2,963 |
4,303 |
6,879 |
59/33 +Probe 20 |
438,708 |
543,560 |
635,481 |
385,704 |
410,210 |
728,374 |
1,263 |
1,475 |
1,151 |
1,295 |
59/52 |
1,058 |
1,196 |
875 |
9,308 |
69,190 |
686 |
926 |
930 |
1,169 |
1,771 |
+Probe 20 |
|
|
|
|
|
|
|
|
|
|
59/33 +Probe 21 |
1,131,775 |
1,206,597 |
957,441 |
1,558 |
260,318 |
653,321 |
1,217 |
3,177 |
1,160 |
2,281 |
59/52 +Probe 21 |
2,260 |
8,505 |
1,529 |
901 |
1,109 |
2,917 |
1,314 |
1,386 |
1,911 |
1,978 |
59/33 +Probe 22 |
475,040 |
486,044 |
474,661 |
342,995 |
713 |
427,613 |
1,257 |
729 |
585 |
1,143 |
59/52 +Probe 22 |
53,691 |
57,052 |
1,205 |
1,225 |
2,526 |
2,948 |
4,203 |
677 |
5,728 |
1,344 |
Table 7.
T7/NT7 + Probe |
P. fal 30 c/rxn |
P. fal 30 c/rxn |
P. fal 30 c/rxn |
P. fal 10 c/rxn |
P. fal 10 c/rxn |
P. fal 10 c/rxn |
B. mic 1e6 c/rxn |
B.mic 1e6 c/rxn |
NEG |
NEG |
66/49 +Probe 7 |
1,697,508 |
1,654,623 |
1,702,775 |
1,632,642 |
1,670,622 |
1,710,421 |
4,352 |
6,899 |
1,718 |
780 |
66/52 +Probe 7 |
698,772 |
9,692 |
1,394,770 |
1,732 |
926 |
198,284 |
3,588 |
4,249 |
1,173 |
890 |
66/33 + Probe 20 |
2,085,094 |
1,917,183 |
1,977,048 |
2,166,352 |
1,898,001 |
1,529,882 |
1,294 |
1,645 |
2,378 |
1,368 |
66/52 + Probe 20 |
5,387 |
6,054 |
1,395,984 |
78,381 |
13,600 |
1,825 |
2,979 |
5,688 |
5,636 |
2,165 |
66/33 + Probe 21 |
2,787,188 |
2,641,943 |
2,691,394 |
2,189,645 |
1,834,205 |
2,528,971 |
5,627 |
3,547 |
3,817 |
4,417 |
66/52 + Probe 21 |
103,016 |
1,944,005 |
8,745 |
2,694,902 |
1,167,241 |
5,856 |
3,229 |
4,437 |
6,515 |
2,704 |
66/33 + Probe 22 |
1,976,930 |
2,048,269 |
2,206,921 |
2,248,244 |
1,576,131 |
1,897,000 |
1,619 |
5,010 |
2,252 |
2,978 |
66/52 + Probe 22 |
314,430 |
1,222,503 |
36,814 |
1,596 |
60,021 |
110,944 |
1,831 |
2,930 |
2,213 |
1,337 |
[0157] Group 3. Re-designs of the non-T7 primer of SEQ ID NO:33 were tested. Each of probes 7 and
8 were paired with each of SEQ ID NO:66/SEQ ID NO:25 and SEQ ID NO 66/SEQ ID NO:35
(T7/NT7). In addition, probe 20 was paired with SEQ ID NO:66/SEQ ID NO:33 (T7/NT7).
[0158] The results of this probe screen are shown in Table 8 below. The T7/NT7 oligomer
pair of SEQ ID NO:66/SEQ ID NO:33 performed well with probe 20. Redesigns for the
non-T7 oligomer of SEQ ID NO:33 (SEQ ID NO:25 and SEQ ID NO:35) induced false positives
with probes 7 and 8.
Table 8.
T7/NT7 + Probe |
P. fal 30 c/rxn |
P. fal 30 c/rxn |
P. fal 30 c/rxn |
P. fal 10 c/rxn |
P. fal 10 c/rxn |
P. fal 10 c/rxn |
NEG |
NEG |
NEG |
NEG |
66/33 +Probe 20 |
2,259,738 |
2,251,254 |
2,259,806 |
2,193,185 |
2,229,852 |
2,305,634 |
10,987 |
2,278 |
2,517 |
833 |
66/25 +Probe 7 |
1,639,345 |
1,634,590 |
1,094,963 |
1,577,109 |
1,447,969 |
1,443,799 |
659,001 |
907,064 |
955,394 |
106,595 |
69/25 +Probe 7 |
1,604,438 |
1,518,505 |
1,606,930 |
1,473,808 |
1,431,398 |
1,542,777 |
873,665 |
963,803 |
849,156 |
705,831 |
66/35 +Probe 7 |
1,590,743 |
1,608,076 |
1,615,693 |
1,595,727 |
1,351,299 |
1,582,092 |
759,553 |
622,876 |
495,724 |
743,057 |
69/35 +Probe 7 |
1,669,062 |
1,684,886 |
1,610,911 |
1,604,953 |
1,279,697 |
1,584,979 |
816,245 |
874,237 |
467,269 |
1,302,044 |
66/25 +Probe 8 |
1,690,077 |
1,673,850 |
1,597,686 |
1,601,896 |
1,359,389 |
1,572,372 |
1,172,325 |
693,937 |
286,919 |
536,015 |
69/25 +Probe 8 |
1,607,570 |
1,637,678 |
1,622,369 |
1,613,782 |
1,538,278 |
1,613,946 |
93,383 |
1,029,827 |
1,213,750 |
1,192,320 |
66/35 +Probe 8 |
1,622,258 |
1,609,624 |
1,572,273 |
1,600,610 |
1,570,232 |
1,611,927 |
1,235,255 |
947,640 |
841,628 |
1,143,229 |
69/35 +Probe 8 |
1,623,329 |
1,679,140 |
1,634,462 |
1,628,953 |
1,509,017 |
1,583,660 |
604,251 |
705,198 |
1,259,683 |
966,102 |
Example 2
[0159] This example describes materials and methods for screening of candidate amplification
systems using TMA on the full automated Procleix Panther system (Grifols Diagnostic
Solutions Inc.).
[0160] Specimens included
P. falciparum, P. knowlesi, P. malariae, P. ovale, and/or
P.
vivax IVT diluted in buffer. Specimens may also include
B. microti IVT as a cross-reactivity specimen. It is necessary to determine that amplification
and detection systems are specific to
Plasmodium. An assay calibrator comprising a
P. falciparum IVT panel at 500 c/mL was included to determine the analyte cutoff for the run. The
assay software uses the analyte cutoff to determine if samples are reactive or non-reactive.
Samples with a signal to cutoff ratio of ≥ 1 are considered reactive, while those
< 1 are non-reactive. Assay Reagents used included the following: a Target Capture
Reagent (TCR) comprising of at least one target capture oligomer (TCO); an Amplification
reagent comprising at least one T7 promoter provider and at least one non-T7 primer;
a Probe reagent consisting of at least one AE labeled probe; Ultrio Plus Enzyme reagent;
and Selection reagent.
Example 3: Plasmodium Target Capture Probe Screening
[0161] Candidate target capture probes (TCOs) were screened with samples containing
P.
falciparum IVT at 500 c/mL prepared in 3 mL Parasite Transport Medium (PTM; 100 mM TRIS, 30
mM magnesium chloride, and 6% (v/v) LLS, at pH 7.5) with 1 mL whole blood. TMA reactions
were performed on the fully automated Procleix Panther system substantially as described
in Example 2. TCOs of SEQ ID NOs. 1-5, 7, 9, and 10 were tested in reactions using
T7 oligomers of SEQ ID NO:66 and SEQ ID NO:69 (5 pmol/rxn each), non-T7 oligomers
of SEQ ID NO:21 and SEQ ID NO:30 (5 pmol/rxn each), and detection probes of SEQ ID
NO:148 and SEQ ID NO: 152 (1.9e6 RLU/rxn each).
[0162] Results are shown in Tables 9 and 10 below. Assay performance was best with TCOs
SEQ ID NO:9 and SEQ ID NO: 10. SEQ ID NO: 3 was not optimal in this assay.
Table 9.
TCO |
Mean Total RLU* |
SEQ ID NO: 3 |
663,034 |
SEQ ID NO:1 |
873,914 |
SEQ ID NO:9 |
1,474,284 |
SEQ ID NO:2 |
985,039 |
SEQ ID NO:10 |
1,455,387 |
SEQ ID NO:7 |
793,219 |
SEQ ID NO:5 |
951,504 |
SEQ ID NO:4 |
952,023 |
* N=10 for all TCOs except SEQ ID NOT (N=20) |
Table 10.
TCO |
%CV Total RLU* |
SEQ ID NO: 3 |
19.2 |
SEQ ID NO:1 |
19.7 |
SEQ ID NO:9 |
2.6 |
SEQ ID NO:2 |
11.2 |
SEQ ID NO:10 |
3.6 |
SEQ ID NO:7 |
28.1 |
SEQ ID NO:5 |
14.4 |
SEQ ID NO:4 |
14.4 |
* N=10 for all TCOs except SEQ ID NOT (N=20) |
Example 4: Analytical Sensitivity - LoD of RNA Copies/mL
[0163] Limit of detection (LoD) for a candidate amplification system was assessed by probit
analysis using
in vitro synthesized transcripts for
P. falciparum, P. knowlesi, P. malariae, P. ovale, and
P. vivax. TMA reactions were performed on the fully automated Procleix Panther system substantially
as described in Example 2. The IVT for each species was serially diluted in buffer
to 100, 30, 10, 3, 1 and 0 copies/mL and tested in 32 replicates for each level per
species. Specimens were tested using TMA on the fully automated Procleix Panther system
(Grifols Diagnostic Solutions Inc.). An assay calibrator comprising a
P. falciparum IVT panel at 500 c/mL was included to determine the analyte cutoff for the run. The
assay software uses the analyte cutoff to determine if samples are reactive or non-reactive.
Samples with a signal to cutoff ratio of ≥ 1 are considered reactive, while those
< 1 are nonreactive. For this assay, the following oligomers were used: T7 oligomers
of SEQ ID NO:66 and SEQ ID NO:69 (5 pmol/rxn each), non-T7 oligomers of SEQ ID NO:21
and SEQ ID NO:30 (5 pmol/rxn each), detection probes of SEQ ID NO:148 and SEQ ID NO:152
(1.9e6 RLU/rxn each), and TCOs of SEQ ID NO:9 and SEQ ID NO:10.
[0164] Results are shown in Table 11 below. Similar LoD values were observed for the five
species tested. 95% LoD ranged from 9.4 to 14.8 copies/mL.
Table 11.
In vitro transcript (N=32) |
50% LoD in Copies/mL (Fiducial Limits) |
95% LoD Copies/mL (Fiducial Limits) |
P. falciparum |
2.1 (1.3 - 2.9) |
9.4 (6.5 - 17.3) |
P. knowlesi |
3.3 (2.2 - 4.5) |
14.8 (10.2 - 27.3) |
P. malariae |
3.3 (2.2 - 4.4) |
13.1 (9.1 - 24.4) |
P. ovale |
2.5 (1.5 - 3.5) |
13.6 (9.0 - 27.8) |
P. vivax |
4.5 (3.4 - 5.7) |
11.6 (8.9 - 17.7) |
Example 5: Analytical Sensitivity LoD of RNA Parasites/mL
[0165] Limit of detection (LoD) for a candidate amplification system was assessed by probit
analysis using cultured parasite-infected cells. Specifically, conditions were tested
using a lysed negative whole blood specimen and diluted cultured
P. falciparum-infected erythrocytes. The cultured sample was received with a known percent parasitemia
and RBC count determined by Fluorescence-Activated Cell Sorting (FACS) to estimate
the concentration (parasite per mL value). Based upon the estimated parasite per mL
value, the sample was diluted in normal negative human whole blood to an estimated
6, 4, 2, 1, and 0.5 parasites per mL. The diluted
Plasmodium-infected whole blood was lysed at a ratio of 0.9 mL of whole blood in 2.7mL of Parasite
Transport Medium (PTM; 100 mM TRIS, 30 mM magnesium chloride, and 6% (v/v) LLS, at
pH 7.5). Specimens were tested using TMA on the fully automated Procleix Panther system
(Grifols Diagnostic Solutions Inc.). An assay calibrator comprising a
P. falciparum IVT panel at 500 c/mL was included to determine the analyte cutoff for the run. The
assay software uses the analyte cutoff to determine if samples are reactive or non-reactive.
Samples with a signal to cutoff ratio of ≥ 1 are considered reactive, while those
< 1 are non-reactive. Assay reagents used included the following: a Target Capture
Reagent (TCR) comprising TCOs of SEQ ID NO:9 and SEQ ID NO:10; an Amplification reagent
comprising T7 oligomers of SEQ ID NO:66 and SEQ ID NO:69 (5 pmol/rxn each), non-T7
oligomers of SEQ ID NO:21 and SEQ ID NO:30 (5 pmol/rxn each); a Probe reagent comprising
AE-labeled detection probes of SEQ ID NO:148 and SEQ ID NO:152 (1.9e6 RLU/rxn each);
Ultrio Plus Enzyme reagent; and Selection reagent.
[0166] Results are shown in Table 12 below. 95% LoD was 2.14 parasites/mL. 480 replicates
each of internal control buffer, PTM, and negative lysate were evaluated with no false
positives (0/1,440).
Table 12.
|
50% LoD in Parasites/mL (Fiducial Limits) |
95% LoD in Parasites/mL (Fiducial Limits) |
P. falciparum |
0.35 (0.14-0.56) |
2.14 (1.38-5.17) |
Example 6: Interference and Cross-reactivity with Babesia
[0167] Interference and cross-reactivity with
Babesia microti (a homologous protozoan) was assessed using
in vitro synthesized transcripts. Testing was performed in TMA reactions performed on the
fully automated Procleix Panther system substantially as described in Example 2, with
P. falciparum IVT dilutions between 100-1 c/mL with and without the addition of
B. microti IVT at 1e6 c/mL. The following oligomers were used: T7 oligomers of SEQ ID NO:71
and SEQ ID NO:72 (5 pmol/rxn each), non-T7 oligomers of SEQ ID NO:34 and SEQ ID NO:53
(5 pmol/rxn each), AE-labeled detection probes of SEQ ID NO: 148 and SEQ ID NO: 157
(1.9e6 RLU/rxn each), and TCOs of SEQ ID NO:9 and SEQ ID NO: 10.
[0168] Results are shown in Tables 13 and 14 below. Comparable reactivity and analyte RLU
was observe for
P. falciparum IVT with and without the presence of
B. microti IVT.
Table 13.
|
P. falciparum (copies) |
100 |
30 |
10 |
3 |
1 |
0 |
% Reactive (N=24) |
Control |
100 |
100 |
100 |
58 |
17 |
0 |
Presence of B. microti |
100 |
100 |
96 |
58 |
17 |
0 |
Table 14.
|
P. falciparum (copies) |
100 |
30 |
10 |
3 |
1 |
0 |
Mean RLU (N=24) |
Control |
2,043,991 |
2,031,427 |
1,993,527 |
1,992,755 |
2,001,796 |
2,518 |
Presence of B. microti |
2,055,344 |
2,029,613 |
1,925,711 |
1,777,978 |
1,815,085 |
855 |
Example 7: Evaluation of Redundant Probes
[0169] Redundant probe combinations were assessed using
in vitro synthesized transcripts. Testing was performed in TMA reactions performed on the
fully automated Procleix Panther system substantially as described in Example 2. The
following oligomers were used: (i) T7 oligomers of SEQ ID NO:71 and SEQ ID NO:72 (5
pmol/rxn each), (ii) non-T7 oligomers of SEQ ID NO:34 and SEQ ID NO:53 (5 pmol/rxn
each), (iii) AE-labeled detection probes of SEQ ID NO:148 (4,5 2MeAE linker) and SEQ
ID NO:157 (1.27e6 RLU/rxn each) with either SEQ ID NO:148 (5,6 2MeAE linker) or SEQ
ID NO:148 (6,7 2MeAE linker), and (iv) TCOs of SEQ ID NO:9 and SEQ ID NO:10.
[0170] Results are shown in Table 15 below. "C1" refers to the probe combination containing
SEQ ID NO:148 (5,6 2MeAE linker); "C2" refers to the probe combination containing
SEQ ID NO: 148 (6,7 2MeAE linker). Comparable reactivity was observed for all
Plasmodium species at 30 and 10 c/mL with the redundant probe combinations. Consistent with
results in Example 1, probes having the same nucleotide sequence (SEQ ID NO:148) but
with different 2MeAE linker sites performed well irrespective of their different labelling.
Table 15.
|
|
P. falciparum (copies) |
P. knowlesi (copies) |
P. malariae (copies) |
P. ovale (copies) |
P. vivax (copies) |
|
|
30 |
10 |
30 |
10 |
30 |
10 |
30 |
10 |
30 |
10 |
% Reactive (N=8) |
C1 |
100 |
88 |
100 |
100 |
100 |
100 |
100 |
75 |
100 |
100 |
C2 |
100 |
75 |
100 |
88 |
100 |
100 |
100 |
88 |
100 |
100 |
Example 8: Analytical Sensitivity - LoD of RNA Parasites/mL
[0171] Limit of detection (LoD) for a candidate amplification system was assessed by probit
analysis using cultured
P. falciparum-infected erythrocytes. Testing was performed in TMA reactions performed on the fully
automated Procleix Panther system substantially as described in Example 5. The following
oligomers were used: (i) T7 oligomers of SEQ ID NO:71 and SEQ ID NO:72 (5 pmol/rxn
each), (ii) non-T7 oligomers of SEQ ID NO:34 and SEQ ID NO:53 (5 pmol/rxn each), (iii)
AE-labeled detection probes of SEQ ID NO:148 (4,5 2MeAE linker), SEQ ID NO:157, and
SEQ ID NO:148 (5,6 2MeAE linker) (1.17e6, 7.92e5, and 2.03e6 RLU/rxn, respectively),
and (iv) TCOs of SEQ ID NO:9 and SEQ ID NO: 10.
[0172] Results are shown in Table 16 below. 95% LoD was 2.31 parasites/mL. There were no
false positives in the negative specimens tested. These results are comparable to
the system evaluated in Example 5 where the 95% LoD was 2.14 parasites/mL.
Table 16.
|
50% LoD in Parasites/mL (Fiducial Limits) |
95% LoD in Parasites/mL (Fiducial Limits) |
P. falciparum |
0.70 (0.33-0.97) |
2.31 (1.61-5.82) |
Example 9
[0173] Primer screening was performed using procedures for a manual Biphasic Real-Time TMA
Assay. For the target capture step, 400 µL of Target Capture Reagent (TCR) comprising
at least 1 Target Capture Oligo (TCO) and 1 T7 promoter provider was added to a 2mL
Deep Well 96-well plate (Thermo Scientific Cat. No. 95040450), followed by 500 µL
of specimen. Specimens consisted of
Plasmodium species
in vitro transcript (IVT) diluted in buffer for
Plasmodium detection and
Babesia species IVT diluted in buffer for
Babesia detection. Specimens may also include
B. microti IVT or
P. falciparum IVT as a cross-reactivity specimen for opposing detection systems due to the conserved
regions between
Babesia and
Plasmodium. It is necessary to determine that amplification and detection systems are specific
to the analyte system. The plate was covered with a sealing card and loaded on to
a Torrey Pines plate incubator and covered with the lid. Incubation steps for the
Torrey Pines incubator included 7 minutes at 80°C followed by 17 minutes at 62°C and
between 15 to 25 minutes at 25°C, respectively.
[0174] After target capture incubation steps, the plate was loaded with a deep well comb
tip (Thermo Scientific Cat. No. 97002534) and placed on to a Kingfisher 96 instrument
(Thermo Scientific Type 710 REF 5400500) fitted with deep well magnets. The Kingfisher
instrument was additionally loaded with a wash plate consisting of a 2mL 96-well plate
(Nunc Deep Well plate Cat. No. 278752) prepared with 500 µL of commercially available
Procleix Wash Buffer reagent (Grifols Diagnostic Solutions Inc.) and a second wash
plate consisting of a 200µL 96-well plate (Thermo Scientific Cat. No. 97002540) containing
200 µL of the wash buffer. For the deep well wash steps, the plate containing the
hybridized TCR-sample mixture was mixed for 5 minutes before collecting magnetic beads
for 20 counts and eluting for 20 seconds to the plate containing 500 µL of wash buffer.
The 500 µL wash plate was mixed for 1 minute before collecting magnetic beads for
10 counts and eluting for 20 seconds to the wash plate containing 200 µL of wash buffer.
[0175] The second wash plate containing the mixture of hybridized magnetic beads and wash
buffer was removed from the Kingfisher instrument, loaded with a small PCR tip comb
(Thermo Scientific 97002514) and transferred to a second Kingfisher 96 instrument
(Thermo Scientific Type 710 REF 5400500) fitted with PCR magnets. The Kingfisher instrument
was additionally loaded with a 96-well PCR plate (Axygen Cat. No. PCR-96-HS-C) containing
30 µL of Amplification Reagent (without Phenol Red) containing at least 1 non-T7 primer
(amplification plate). To transfer the hybridized magnetic beads to the amplification
plate the wash plate was mixed for 5 minutes before collecting magnetic beads for
30 counts and eluting for 30 seconds to the amplification plate. The wash plate was
mixed again for 1 minute before collecting magnetic beads for 30 counts and eluting
for 30 seconds to the amplification plate to complete the transfer.
[0176] The amplification plate was covered with a sealing card and loaded on to a Stratagene
instrument (Mx3005P Multiplex Quantitative PCR System) to incubate for 5 minutes at
43°C. The plate was transferred to a heat block set to 42°C and uncovered to add 10
µL of commercially available Ultrio Plus Enzyme reagent (Grifols Diagnostic Solutions
Inc.) and re-covered with a sealing card. The plate was mixed on the heat block for
1 minute at 1400 RMP and reloaded on the Stratagene instrument to incubate for 5 minutes
at 43°C. The plate was transferred again to the heat block and uncovered to add 15
µL of Promoter reagent (Amplification Reagent without Phenol Red) containing a mixture
of at least 1 T7 promoter provider and at least 1 Fluorescent labeled molecular Torch
or Beacon (5'-Hexochloro-Fluorescein (HEX) for
Plasmodium or 5'-Fluorescein (FAM) for
Babesia) and sealed with a clear adhesive plate cover. The plate was mixed on the heat block
for 1 minute at 1400 RMP and reloaded on the Stratagene for the read protocol. The
read protocol consisted of incubation at 43°C and read every 30 seconds for cycles
of 120 or 150.
[0177] The raw data exported from the Stratagene instrument was analyzed using an inhouse
software tool. Fluorescent curves were analyzed using a threshold of 1,000 Relative
Fluorescent Units (RFU). The time for specimens to meet or exceed the threshold (TTime)
was determined by the software. Specimens with a TTime were considered Reactive for
Plasmodium in the HEX channel or
Babesia in the FAM channel. Lower TTimes indicated better performance of the tested systems.
Specimens with no TTime, or under the threshold, were considered non-reactive.
[0178] In this example, combinations of molecular torch or beacon probes and amplification
oligomer pairs were tested in three groups (Groups 1, 2, and 3). Tested oligomer combinations
are shown in Table 17 below.
Table 17. Combinations of Amplification Oligomers and Torch Probes.
T7 Primer |
Non-T7 Primer |
Probe (Torch/Beacon) |
Group |
SEQ ID NO:181 |
SEQ ID NO:183 |
SEQ ID NO:104 (torch) |
Group 1 |
SEQ ID NO:181 |
SEQ ID NO:183 |
SEQ ID NO:105 (torch) |
SEQ ID NO:181 |
SEQ ID NO:183 |
SEQ ID NO:106 (torch) |
SEQ ID NO:181 |
SEQ ID NO:183 |
SEQ ID NO:121 (torch) |
SEQ ID NO:181 |
SEQ ID NO:183 |
SEQ ID NO:107 (beacon) |
SEQ ID NO:181 |
SEQ ID NO:183 |
SEQ ID NO:108 (beacon) |
SEQ ID NO:181 |
SEQ ID NO:184 |
SEQ ID NO:104 (torch) |
Group 2 |
SEQ ID NO:59 |
SEQ ID NO:51 |
SEQ ID NO:123 (torch) |
Group 3 |
SEQ ID NO:59 |
SEQ ID NO:31 |
SEQ ID NO:123 (torch) |
SEQ ID NO:59 |
SEQ ID NO:50 |
SEQ ID NO:123 (torch) |
[0179] Group 1 Results. Probes of SEQ ID NOs: 104-107 and 121 successfully detected
Plasmodium with fluorescent curves generating average TTimes of less than or equal to 30.69
minutes at 10 c/mL. No fluorescent curves with TTimes were generated for the probe
of SEQ ID NO: 108 concluding it was unable to detect
Plasmodium.
[0180] Group 2 Results. Amplification oligomers of SEQ ID NOs:181 and 184 in combination with the torch probe
of SEQ ID NO:104 successfully detected
Plasmodium, demonstrated by fluorescent curves generating TTimes of less than or equal to 20.89
minutes at 10 c/mL, and showed no cross-reactivity with
Babesia IVT, demonstrated by the absence of a fluorescent curve with a TTime.
[0181] Group 2 Results. Non-T7 amplification oligomers of SEQ ID NOs:51, 31, and 50 each in combination with
the T7 amplification oligomer of SEQ ID NO:59 and the torch probe of SEQ ID NO:123
successfully detected
Plasmodium as demonstrated by fluorescent curves generating TTimes of less than or equal to
22.82 minutes.
Example 10
[0182] A candidate amplification system was tested using Real-time TMA on the fully automated
Panther system installed with Real-time Fluorometers (Hologic Inc.). Specimens consisted
of
Plasmodium species IVT diluted in buffer for
Plasmodium detection and
Babesia species IVT diluted in buffer for
Babesia detection. Specimens may also include
B. microti IVT or
P. falciparum IVT as a cross reactivity specimen. It is necessary to determine that amplification
and detection systems are specific to the analyte system. Assay Reagents used included
the following: a TCR comprising of at least one TCO and one T7 promoter provider;
an Amplification Reagent comprising of at least one non-T7 primer; a Promoter reagent
(Amplification Reagent without Phenol Red) consisting of at least one T7 promoter
provider and at least one Fluorescent labeled molecular Torch or Beacon (5'-Hexochloro-Fluorescein
(HEX) for Plasmodium or 5'-Fluorescein (FAM) for
Babesia); and Ultrio Plus Enzyme reagent.
[0183] For analysis of the raw data exported from the Panther system, the Panther RT-Dev
Tool (Hologic Inc.) was used. Fluorescent curves were analyzed using a threshold of
1,000 Relative Fluorescent Units (RFU). The time for specimens to meet or exceed the
threshold (TTime) was determined by the software. Specimens with a TTime were considered
Reactive for
Plasmodium in the HEX channel or
Babesia in the FAM channel. Lower TTimes indicated better performance of the tested systems.
Specimens with no TTime, or under the threshold, were considered non-reactive.
[0184] The oligomers tested in this example were a T7 oligomer of SEQ ID NO:59, a non-T7
oligomer of SEQ ID NO:30, and a torch probe of SEQ ID NO: 123. Results demonstrated
detection of 8 out of 8 replicates tested at 30 c/mL of
Plasmodium IVT and 3 out of 8 replicates tested at 10 c/mL. Fluorescent curves generated TTimes
of less than or equal to 33.18 minutes for the detected replicates at 10 c/mL.
Example 11: Detection of Plasmodium in Clinical Specimens
[0185] Candidate amplification systems were tested using Real-time TMA on the fully automated
Panther system installed with Real-time Fluorometers (Hologic Inc.). Specimens consisted
of positive controls for
B. microti and
P. falciparum IVT diluted in buffer to 300 c/mL and a negative control consisting of negative buffer.
Clinical specimens consisted of
P. falciparum and
P. ovale whole blood and plasma specimens. Whole blood specimens were prepared by manual addition
of 100 µL to 3 mL of a lysis buffer (14 mM sodium bicarbonate, 250 mM ammonium chloride,
5% (v/v) LLS, and 0.1 mM EDTA, at a pH of 7.4). Plasma specimens were prepared by
manual addition of 100 µL to 3 mL of processed human plasma. Specimens were tested
on the Panther system. Assay Reagents used included the following: a TCR comprising
of at least one TCO and one T7 promoter provider; an Amplification Reagent comprising
of at least one non-T7 primer; a Promoter reagent (Amplification Reagent without Phenol
Red) consisting of at least one T7 promoter provider and at least one Fluorescent
labeled molecular Torch or Beacon (5'-Hexochloro-Fluorescein (HEX) for
Plasmodium or 5'-Fluorescein (FAM) for
Babesia); and Enzyme reagent.
[0186] For analysis of the raw data exported from the Panther system, the Panther RT-Dev
Tool (Hologic Inc.) was used. Fluorescent curves were analyzed using a threshold of
1,000 Relative Fluorescent Units (RFU). The time for specimens to meet or exceed the
threshold (TTime) was determined by the software. Specimens with a TTime were considered
Reactive for
Plasmodium in the HEX channel or
Babesia in the FAM channel Lower TTimes indicated better performance of the tested systems.
Specimens with no TTime, or under the threshold, were considered Nonreactive.
[0187] The oligomers tested in this example were a T7 oligomer of SEQ ID NO:59, a non-T7
oligomer of SEQ ID NO:30, and a torch probe of SEQ ID NO:123. Results are shown in
Table 18 below. Real-time TMA results with the tested oligomers showed 100% concordance
with PCR results. Based on the calibration curve generated using
in vitro Plasmodium falciparum infected erythrocytes, the tested sample have between 7.14E6 and 2.14E8 parasites/mL
in whole blood. No cross reactivity was observed with
Babesia.
Table 18. Detection of Plasmodium positive Clinical Specimens.
|
|
Whole Blood |
Plasma |
Whole Blood |
Plasma |
|
Sample |
Organism |
PCR Result (Wadsworth) |
Real-time TMA #reactive/ #tested |
Real-time TMA #reactive/ #tested |
Est. # par/mL |
Est. # par/mL |
Relative amounts of parasites (WB/plasma) |
1 |
P. fal |
Positive |
4/4 |
4/4 |
2.14E+08 |
3.18E+02 |
672,841 |
2 |
P. fal |
Positive |
4/4 |
4/4 |
1.49E+08 |
2.63E+02 |
566,724 |
3 |
P. fal |
Positive |
4/4 |
4/4 |
1.11E+08 |
2.55E+03 |
43,685 |
4 |
P. fal |
Positive |
4/4 |
4/4 |
7.28E+07 |
4.60E+02 |
158,340 |
5 |
P. fal |
Positive |
4/4 |
4/4 |
5.49E+07 |
1.08E+02 |
508,948 |
6 |
P. fal |
Positive |
4/4 |
4/4 |
3.07E+07 |
1.96E+01 |
1,568,437 |
7 |
P. fal |
Positive |
4/4 |
4/4 |
2.27E+07 |
2.22E+01 |
1,026,604 |
8 |
P. fal |
Positive |
4/4 |
4/4 |
1.09E+07 |
2.38E+01 |
457,997 |
9 |
P. ova |
Positive |
4/4 |
4/4 |
1.05E+07 |
3.21E+04 |
329 |
10 |
P. fal |
Positive |
4/4 |
4/4 |
7.14E+06 |
1.74E+01 |
411,025 |
Example 12
[0188] Candidate amplification systems were tested using Real-time TMA on the fully automated
Panther system installed with Real-time Fluorometers (Hologic Inc.). Specimens consisted
of 5 strains of
Plasmodium infected erythrocytes: US 05 F Benin I, US 05 F Santa Lucia, US 08 F Nigeria XII,
US05 F FC27/A3, and US 05 F PH1. Infected erythrocytes were provided with an estimated
parasite/mL value. Each strain was serially diluted in normal negative human whole
blood to estimated values of 10 parasites/mL. The diluted
Plasmodium-infected whole blood was manually lysed at a ratio of 1 mL of whole blood in 3 mL
of a lysis buffer (14 mM sodium bicarbonate, 250 mM ammonium chloride, 5% (v/v) LLS,
and 0.1 mM EDTA, at a pH of 7.4). Specimens were tested on the Panther system. Assay
Reagents used included the following a TCR comprising of at least one TCO and one
T7 promoter provider; an Amplification Reagent comprising of at least one non-T7 primer;
a Promoter reagent (Amplification Reagent without Phenol Red) consisting of at least
one T7 promoter provider and at least one Fluorescent labeled molecular Torch or Beacon
(5'-Hexochloro-Fluorescein (HEX) for Plasmodium or 5'-Fluorescein (FAM) for
Babesia); and Enzyme reagent.
[0189] For analysis of the raw data exported from the Panther system, the Panther RT-Dev
Tool (Hologic Inc.) was used. Fluorescent curves were analyzed using a threshold of
1,000 Relative Fluorescent Units (RFU). The time for specimens to meet or exceed the
threshold (TTime) was determined by the software. Specimens with a TTime were considered
Reactive for
Plasmodium in the HEX channel or
Babesia in the FAM channel. Lower TTimes indicated better performance of the tested systems.
Specimens with no TTime, or under the threshold, were considered Nonreactive.
[0190] The oligomers tested in this example were a T7 oligomer of SEQ ID NO:59, a non-T7
oligomer of SEQ ID NO:30, and a torch probe of SEQ ID NO:123. Results demonstrated
that all 5 strains of
Plasmodium infected erythrocytes were detected in 6 out of 6 replicates tested at 10 parasites/mL
with fluorescent curves generating TTimes ranging from 14.29 to 17.17 minutes.
Table 19. Exemplary Sequences.
SEQ ID NO: |
Sequence (5' to 3') |
Comments |
1 |
 |
Target capture oligomer |
2 |
 |
Target capture oligomer |
3 |
 |
Target capture oligomer |
4 |
 |
Target capture oligomer |
5 |
 |
Target capture oligomer |
6 |
 |
Target capture oligomer |
7 |
 |
Target capture oligomer |
8 |
 |
Target capture oligomer |
9 |
 |
Target capture oligomer |
10 |
 |
Target capture oligomer |
11 |
GGAUUGGGUAAUUUGCGCGCCC |
THS of SEQ ID NO: 1 |
12 |
CAAGAAAGAGCUAUCAAUCUGUCAAUCC |
THS of SEQ ID NO:2 |
13 |
CCCGUGUUGAGUCAAAUUAAGCCGCA |
THS of SEQ ID NO:3 |
14 |
GGGUAAUUUGCGCGCCUGCUGC |
THS of SEQ ID NO:4 |
15 |
UUUCUCAGGCUCCCUCUCCGGAAUCG |
THS of SEQ ID NO:5 |
16 |
ACAUCUGAAUACGAAUGUCCCCAA |
THS of SEQ ID NO:6 |
17 |
CUAGUCGGCAUAGUUUAUGGUUA |
THS of SEQ ID NO:7 |
18 |
AAAAACGGCCAUGCAUCACCAUCC |
THS of SEQ ID NO:8 |
19 |
UAGGCCAAUACCCUACCGUCC |
THS of SEQ ID NO:9 |
20 |
AAAGACUUUGAUUUCUCUCAAGG |
THS of SEQ ID NO: 10 |
21 |
AATACTACAGCATGG |
Non-T7 amp oligo |
22 |
GGAAGGCAGCAGGCGCGTA |
Non-T7 amp oligo |
23 |
AATACTACAGCATGGA |
Non-T7 amp oligo |
24 |
AATACTACAGCATGGAA |
Non-T7 amp oligo |
25 |
ATACTACAGCATGGAATA |
Non-T7 amp oligo |
26 |
ATTCAGATGTCAGAGGTGA |
Non-T7 amp oligo |
27 |
GTATTCAGATGTCAGAGGTGA |
Non-T7 amp oligo |
28 |
GTTACGATTAATAGGAGT |
Non-T7 amp oligo |
29 |
GTTACGATTAATAGGAGTA |
Non-T7 amp oligo |
30 |
GTTACGATTAATAGGAGTAG |
Non-T7 amp oligo |
31 |
GTTACGATTAATAGGAGTAGC |
Non-T7 amp oligo |
32 |
AATACTACAGCATGGAAT |
Non-T7 amp oligo |
33 |
AATACTACAGCATGGAATA |
Non-T7 amp oligo |
34 |
TACGATTAATAGGAGT |
Non-T7 amp oligo |
35 |
TACTACAGCATGGAATA |
Non-T7 amp oligo |
36 |
TATTCAGATGTCAGAGGTGA |
Non-T7 amp oligo |
37 |
TCAGTNCCTTATGAGAAATC |
Non-T7 amp oligo |
38 |
TGGCTTAGTTACGATT |
Non-T7 amp oligo |
39 |
TGGCTTAGTTACGATTAATAG |
Non-T7 amp oligo |
40 |
TTAATAGGAGTAGCTTGGGG |
Non-T7 amp oligo |
41 |
TTACGATTAATAGGAGT |
Non-T7 amp oligo |
42 |
TTCAGATGTCAGAGGTGA |
Non-T7 amp oligo |
43 |
TTGGCTTAGTTACGAT |
Non-T7 amp oligo |
44 |
TTGGCTTAGTTACGATTA |
Non-T7 amp oligo |
45 |
TTGGGGACATTCGTATTCAGA |
Non-T7 amp oligo |
46 |
TTTAGATTGCTTCCTTCAGT |
Non-T7 amp oligo |
47 |
TTTGAATACTANAGCA |
Non-T7 amp oligo |
48 |
ACATTCGTATTCAGATGTCAG |
Non-T7 amp oligo |
49 |
CTTAGTTACGATTAATAGGA |
Non-T7 amp oligo |
50 |
CGATTAATAGGAGTAGCTTGG |
Non-T7 amp oligo |
51 |
CTTAGTTACGATTAATAGGAGTAG |
Non-T7 amp oligo |
52 |
CTTGAATACTNCAGCA |
Non-T7 amp oligo |
53 |
GGCTTAGTTACGATTA |
Non-T7 amp oligo |
54 |
AATACTANAGCATGG |
Non-T7 amp oligo |
55 |
AATACTANAGCATGGAATA |
Non-T7 amp oligo |
56 |
AATTCTAAAGAAGAGAG |
Non-T7 amp oligo |
57 |
 |
T7 amp oligo |
58 |
 |
T7 amp oligo |
59 |
 |
T7 amp oligo |
60 |
 |
T7 amp oligo |
61 |
 |
T7 amp oligo |
62 |
 |
T7 amp oligo |
63 |
 |
T7 amp oligo |
64 |
 |
T7 amp oligo |
65 |
 |
T7 amp oligo |
66 |
 |
T7 amp oligo |
67 |
 |
T7 amp oligo |
68 |
 |
T7 amp oligo |
69 |
 |
T7 amp oligo |
70 |
 |
T7 amp oligo |
71 |
 |
T7 amp oligo |
72 |
 |
T7 amp oligo |
73 |
 |
T7 amp oligo |
74 |
 |
T7 amp oligo |
75 |
 |
T7 amp oligo |
76 |
 |
T7 amp oligo |
77 |
 |
T7 amp oligo |
78 |
 |
T7 amp oligo |
79 |
 |
T7 amp oligo |
80 |
TTCACTCCCTTAACTTTCGTTCTTG |
THS of SEQ ID NO:57 |
81 |
CTTGATTAATGGAAGTATTTTAGA |
THS of SEQ ID NO:58 |
82 |
CTTAACTTTCGTTCTTGATTAATGGAAGT |
THS of SEQ ID NO:59 |
83 |
CCTACTCTTGTCTTAAACTA |
THS of SEQ ID NO:60 |
84 |
AAACGGCCATGCATCACCATCCAAGA |
THS of SEQ ID NO:61 |
85 |
CTCCCTTAACTTTCGTTCTTGATTAATGGAAGT |
THS of SEQ ID NO:62 |
86 |
CGACGGTATCTGATCGTCTTCACTCCC |
THS of SEQ ID NO:63 |
87 |
CTTAACTTTCGTTCTTGATTAATGGAAG |
THS of SEQ ID NO:64 |
88 |
CTTAACTTTCGTTCTTGATTAATGGAAGTA |
THS of SEQ ID NO:65 |
89 |
CACTCCCTTAACTTTCGTTCTTGATTAATG |
THS of SEQ ID NO:66 |
90 |
CACTCCCTTAACTTTCGTTCTTGATTAATGG |
THS of SEQ ID NO:67 |
91 |
CTTCACTCCCTTAACTTTCGTTCTTGATT |
THS of SEQ ID NO:68 |
92 |
CTTCACTCCCTTAACTTTCGTTCTTGAT |
THS of SEQ ID NO:69 |
93 |
ATCGTCTTCACTCCCTTAACTTTCGTTC |
THS of SEQ ID NO:70 |
94 |
CTCCCTTAACTTTCGTTCTTGATTAATG |
THS of SEQ ID NO:71 |
95 |
TCACTCCCTTAACTTTCGTTCTTGAT |
THS of SEQ ID NO:72 |
96 |
CCCTTAACTTTCGTTCTTGATTAATG |
THS of SEQ ID NO:73 |
97 |
CTTAACTTTCGTTCTTGATTAATG |
THS of SEQ ID NO:74 |
98 |
TAACTTTCGTTCTTGATTAATG |
THS of SEQ ID NO:75 |
99 |
ACTCCCTTAACTTTCGTTCTTGAT |
THS of SEQ ID NO:76 |
100 |
TCCCTTAACTTTCGTTCTTGAT |
THS of SEQ ID NO:77 |
101 |
AGGCAAATGCTTTCGCAGTTGTTNGTCT |
THS of SEQ ID NO:78 |
102 |
AGGCAAATGCTTTCGCAGTTGTTTGTCT |
THS of SEQ ID NO:79 |
103 |
CGCGCAAGCGAGAAAGCGCG |
Torch detection probe |
104 |
GCUCGCAUUCGCGCAAGCGAGC |
Torch detection probe |
105 |
GCUUGCGAGUAUUCGCGCAAGC |
Torch detection probe |
106 |
GGCAAGCGAGAAAGUCUUGCC |
Torch detection probe |
107 |
CCGAGGUAUUCGCGCAACUCGG |
Beacon detection probe |
108 |
GGCUCACUUUCUCGCUUGGAGCC |
Beacon detection probe |
109 |
CUCUGGAGACNAGCACCAGAG |
Torch detection probe |
110 |
GUCUCAUUUUCUGGAGAC |
Torch detection probe |
111 |
GCCUAAAAUACUUCCUAGGC |
Torch detection probe |
112 |
CAUGGAAAUACUUCCAUG |
Torch detection probe |
113 |
CCGAGAUUUUCUGGAGACCUCGG |
Beacon detection probe |
114 |
CCGAGGCCUAAAAUACUUCCCUCGG |
Beacon detection probe |
115 |
GGGAAUUUAAAACCUUCCC |
Torch detection probe |
116 |
GGAAGGAAUUUAAAACCUUCC |
Torch detection probe |
117 |
GUGGGAAUUUAAAACCCCCAC |
Torch detection probe |
118 |
UCCAGAAAUUCUUAGAUUUUCUGGA |
Torch detection probe |
119 |
ACUCCGAACGAAAGUUAAGGGAGU |
Torch detection probe |
120 |
AGGGAGUGAAGACGAUCAUCCCU |
Torch detection probe |
121 |
UCGCGCAAGCGAGAAAGGCGCGA |
Torch detection probe |
122 |
CCGAAGUGNCUAAAAUACUUCGG |
Torch detection probe |
123 |
CACCUCAGAUGUCAGAGGUG |
Torch detection probe |
124 |
CUACCUCUAAAGAAGAGAGGUAG |
Torch detection probe |
125 |
CGCGCAAGCGAGAAAG |
THS of SEQ ID NO:103 |
126 |
AUUCGCGCAAGCGAGC |
THS of SEQ ID NO:104 |
127 |
GAGUAUUCGCGCAAGC |
THS of SEQ ID NO:105 |
128 |
GGCAAGCGAGAAAGU |
THS of SEQ ID NO: 106 |
129 |
GUAUUCGCGCAA |
THS of SEQ ID NO: 107 |
130 |
ACUUUCUCGCUUG |
THS of SEQ ID NO:108 |
131 |
CUCUGGAGACNAGCA |
THS of SEQ ID NO: 109 |
132 |
AUUUUCUGGAGAC |
THS of SEQ ID NO:110 |
133 |
GCCUAAAAUACUUCC |
THS of SEQ ID NO:111 |
134 |
AAAUACUUCCAUG |
THS of SEQ ID NO:112 |
135 |
AUUUUCUGGAGAC |
THS of SEQ ID NO:113 |
136 |
CCUAAAAUACUUC |
THS of SEQ ID NO:114 |
137 |
GGGAAUUUAAAACC |
THS of SEQ ID NO: 115 |
138 |
GGAAUUUAAAACCUUCC |
THS of SEQ ID NO:116 |
139 |
GUGGGAAUUUAAAACC |
THS of SEQ ID NO:117 |
140 |
GAAAUUCUUAGAUUUUCUGGA |
THS of SEQ ID NO: 118 |
141 |
GAACGAAAGUUAAGGGAGU |
THS of SEQ ID NO:119 |
142 |
AGGGAGUGAAGACGAUCA |
THS of SEQ ID NO: 120 |
143 |
UCGCGCAAGCGAGAAAG |
THS of SEQ ID NO:121 |
144 |
UGNCUAAAAUACUUCGG |
THS of SEQ ID NO: 122 |
145 |
CAGAUGUCAGAGGUG |
THS of SEQ ID NO:123 |
146 |
UCUAAAGAAGAGAGGUAG |
THS of SEQ ID NO: 124 |
147 |
UCUUAGAUUUUCUGGAGAC |
Detection probe |
148 |
UUCAGAUGUCAGAGG |
Detection probe |
149 |
UUCAGAUGUCAGAGGT |
Detection probe |
150 |
UAUUCAGAUGUCAGAGGT |
Detection probe |
151 |
UAUUCAGAUGUCAGAGGUG |
Detection probe |
152 |
UCAGAUGUCAGAGGT |
Detection probe |
153 |
UUCAGAUGUCAGAGGT |
Detection probe |
154 |
AUUCAGAUGUCAGAGGT |
Detection probe |
155 |
AUUCAGAUGUCAGAGGUG |
Detection probe |
156 |
CUUAGAUUUUCUGGAGA |
Detection probe |
157 |
CUUAGAUUUUCUGGAGAC |
Detection probe |
158 |
CUUAGUUACGAUUAAUAGGA |
Detection probe |
159 |
GUAUUCAGAUGUCAGAGGUGA |
Detection probe |
160 |
AUUCUUAGAUUUUCUGGAGAC |
Detection probe |
161 |
CUAAGAUUUUCUGGAGAC |
Detection probe |
162 |
TTTGAATACTANAGCATGGAATA |
Amp oligo hybridizing region (SEQ ID NOs:21, 23-25, 32, 33, 35, 54, & 55 are contained
within here) |
163 |
TACTANAGCA |
Amp oligo core sequence (SEQ ID NOs:21, 23-25, 32, 33, 35, 54, & 55 share this) |
164 |
TTTGAATACTACAGCATGGAATA |
Amp oligo hybridizing region (SEQ ID NOs:21, 23-25, 32, 33, & 35 are contained within
here) |
165 |
TACTACAGCA |
Amp oligo core sequence (SEQ ID NOs:21, 23-25, 32, 33, & 35 share this) |
166 |
TTGGCTTAGTTACGATTAATAGGAGTAGCTTGGGG |
Amp oligo hybridizing region (SEQ ID NOs:28-31, 34, 38-41, 43, 44, 49-51, & 53 are
contained within here) |
167 |
TTAATAGGAGT |
Amp oligo core sequence (SEQ ID NOs:28-31, 34, 40, 41, & 49-51 share this) |
168 |
GGCTTAGTTACGAT |
Amp oligo core sequence (SEQ ID NOs:38, 39, 43, 44, & 53 share this) |
169 |
 |
Amp oligo hybridizing region (SEQ ID NOs:80-82, & 85-100 are contained within here) |
170 |
 |
Amp oligo hybridizing region (SEQ ID NOs:80-82, 85, & 87-100 are contained within
here) |
171 |
CTTGATTAATG |
Amp oligo core sequence (SEQ ID NOs:81, 82, 85, 87-90, 94, & 96-98 share this) |
172 |
TAACTTTCGTTC |
Amp oligo core sequence (SEQ ID NOs:80, 82, 85, & 87-100 share this) |
173 |
CTTCACTCCC |
Amp oligo core sequence (SEQ ID NOs:86 & 91-93 share this) |
174 |
UCAGAUGUCAGAGG |
Detection probe core sequence (SEQ ID NOs: 148-155 & 159 share this) |
175 |
CUAAGAUUUUCUGGAGA |
Detection probe core sequence (SEQ ID NOs: 147, 156, 157, 160, & 161 share this) |
176 |
CAGAUGUCAGAGG |
Detection probe core sequence (SEQ ID NOs: 145, 148-155, & 159 share this) |
177 |
YUCUGGAGAC |
Detection probe core sequence (SEQ ID NOs: 131, 132, 135, 147, 156, 157, 160, & 161
share this) |
178 |
AUUUUCUGGA |
Detection probe core sequence (SEQ ID NOs: 132, 135, 140, 147, 156, 157, 160, & 161
share this) |
179 |
AATTTAATACGACTCACTATAGGGAGA |
Exemplary T7 promoter sequence for use in an isothermal amplification reaction |
180 |
 |
Plasmodium falciparum 3D7 18S ribosomal RNA (PF3D7 _0725600), rRNA |
NCBI Reference Sequence: XR_002273081.2 |
|
 |
|
181 |
 |
T7 amp oligo |
182 |
TCAAGAAAGAGCTATNAATCTGTCAATCC |
THS of SEQ ID NO:181 |
183 |
GAAATCAAAGTCTTTGGGTTCTG |
Non-T7 amp oligo |
184 |
CAAAGTCTTTGGGTTCTGG |
Non-T7 amp oligo |
185 |
 |
Amp oligo hybridizing region (SEQ ID NOs:37, 46, 183, & 184 are contained within here) |
186 |
GAAATCAAAGTCTTTGGGTTCTGG |
Amp oligo hybridizing region (SEQ ID NOs: 183 & 184 are contained within here) |
187 |
CAAAGTCTTTGGGTTCTG |
Amp oligo core sequence (SEQ ID NOs: 183 & 184 share this) |
188 |
 |
Amp oligo hybridizing region (SEQ ID NOs:83, 84, & 182 are contained within here) |
189 |
GAGUAUUCGSGCAAGCGAGAAAGU |
Detection probe hybridizing region (SEQ ID NOs:125-130 & 143 are contained within
here) |
190 |
AUUCGCGCAA |
Detection probe core sequence (SEQ ID NOs: 126, 127, & 129 share this) |
191 |
CAAGCGAGC |
Detection probe core sequence (SEQ ID NOs: 125, 126, 128, 130, & 143 share this) |
192 |
[GenBank Accession JQ627153.1] |
Plasmodium vivax isolate SV1 18S ribosomal RNA gene, partial sequence |
193 |
[GenBank Accession L07560.1] |
Plasmodium knowlesi small subunit ribosomal RNA sequence |
194 |
[GenBank Accession AB182491.1] |
Plasmodium ovale gene for small subunit ribosomal RNA, complete sequence, variant
type 1 |
195 |
[GenBank Accession AF487999.1] |
Plasmodium cf. malariae type 1 small subunit ribosomal RNA gene, complete sequence |
196 |
 |
Detection probe hybridizing region (SEQ ID NOs:131, 132, 135, 140, 145, 147-157 &
159-161 are contained within here) |
197 |
 |
Detection probe hybridizing region (SEQ ID NOs:132, 135, 140, 145, 147-157 & 159-161
are contained within here) |