(19)
(11)EP 2 753 629 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
19.10.2016 Bulletin 2016/42

(21)Application number: 12829439.4

(22)Date of filing:  06.09.2012
(51)Int. Cl.: 
C07H 1/08  (2006.01)
C12N 15/10  (2006.01)
C12Q 1/68  (2006.01)
(86)International application number:
PCT/US2012/053909
(87)International publication number:
WO 2013/036603 (14.03.2013 Gazette  2013/11)

(54)

METHODS FOR DETECTING LYME DISEASE

VERFAHREN FÜR DEN NACHWEIS VON LYME-KRANKHEIT

PROCÉDÉ POUR DÉTECTER LA MALADIE DE LYME


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 06.09.2011 US 201161531471 P

(43)Date of publication of application:
16.07.2014 Bulletin 2014/29

(73)Proprietor: Ibis Biosciences, Inc.
Carlsbad, CA 92008 (US)

(72)Inventors:
  • ESHOO, Mark W.
    San Diego, California 92130 (US)
  • CROWDER, Christopher
    San Marcos, California 92078 (US)

(74)Representative: Chapman, Desmond Mark 
Carpmaels & Ransford LLP One Southampton Row
London WC1B 5HA
London WC1B 5HA (GB)


(56)References cited: : 
US-A1- 2003 054 338
US-A1- 2010 190 240
US-A1- 2006 078 923
  
  • D. LIVERIS ET AL: "Improving the Yield of Blood Cultures from Patients with Early Lyme Disease", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 49, no. 6, 13 April 2011 (2011-04-13) , pages 2166-2168, XP055181851, ISSN: 0095-1137, DOI: 10.1128/JCM.00350-11
  • VAN DAM ALJE P: "Molecular diagnosis of Borrelia bacteria for the diagnosis of Lyme disease.", EXPERT OPINION ON MEDICAL DIAGNOSTICS MAR 2011, vol. 5, no. 2, March 2011 (2011-03), pages 135-149, XP009183653, ISSN: 1753-0059
  • BABADY N E; PATEL R; SLOAN L M; VETTER E A; SPURBECK D K; BINNICKER M J: "Positivity Rate for Detection of Borrelia burgdorferi in Blood, Cerebrospinal Fluid, Synovial Fluid and Tissue by Real-Time PCR", ABSTRACTS OF THE GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY., vol. 108, 1 June 2008 (2008-06-01), XP055181911, US ISSN: 1060-2011
  • LIVERIS D ET AL: "Quantitation of cell-associated borrelial DNA in the blood of Lyme disease patients with erythema migrans", EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY & INFECTIOUS DISEASES, SPRINGER, BERLIN, DE, vol. 31, no. 5, 16 August 2011 (2011-08-16), pages 791-795, XP035039694, ISSN: 1435-4373, DOI: 10.1007/S10096-011-1376-X
  • FOMENKO N V ET AL: "Diversity of Borrelia burgdorferi sensu lato in natural foci of Novosibirsk region", INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, URBAN UND FISCHER, DE, vol. 298, 1 September 2008 (2008-09-01), pages 139-148, XP023520152, ISSN: 1438-4221, DOI: 10.1016/J.IJMM.2007.11.008 [retrieved on 2008-01-30]
  • E. POSTNIKOVA ET AL: "Identification of Bacterial Plant Pathogens Using Multilocus Polymerase Chain Reaction/Electrospray Ionization-Mass Spectrometry", PHYTOPATHOLOGY, vol. 98, no. 11, 1 November 2008 (2008-11-01), pages 1156-1164, XP055182137, ISSN: 0031-949X, DOI: 10.1094/PHYTO-98-11-1156
  • HOFSTADLER S A: "High throughput characterization of amplified nucleic acids by ESI-TOF mass spectrometry: Applications in pathogen detection and characterization", MOLECULAR & CELLULAR PROTEOMICS, vol. 6, no. 8, Suppl. S, August 2007 (2007-08), page 66, XP055182186, & 8TH INTERNATIONAL SYMPOSIUM ON MASS SPECTROMETRY IN THE HEALTH AND LIFE SCIENCES; SAN FRANCISCO, CA, USA; AUGUST 18 -23, 2007 ISSN: 1535-9476
  • MARK W. ESHOO ET AL: "Direct Molecular Detection and Genotyping of Borrelia burgdorferi from Whole Blood of Patients with Early Lyme Disease", PLOS ONE, vol. 7, no. 5, 8 May 2012 (2012-05-08), page e36825, XP055182231, DOI: 10.1371/journal.pone.0036825
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

FIELD OF THE INVENTION



[0001] The present invention provides methods of detecting Lyme disease comprising whole blood nucleic acid extraction methods, as well as nested isothermal amplification methods. In certain embodiments, these methods are applied to detecting Lyme disease in patients without classic erythema migrans skin lesions.

BACKGROUND



[0002] The diagnosis of acute infectious diseases is problematic when the pathogens are not in abundance, not easily cultured or the antibody response to them is delayed. Lyme disease is such an example. Infection by the causative agent, Borrelia burgdorferi1,2, is often difficult to diagnose because of clinical variability, including variations of the erythema migrans (EM) skin lesion and the lack of high performing diagnostic tests. A strategy that results in early unambiguous diagnosis of the infection will favorably impact management of the patient.

[0003] There are currently clinical limitations to early diagnosis. The best clinical marker of acute infection, the skin lesion erythema migrans (EM), is absent in at least 30% of the cases.3 Even when present, the EM is often not the classic bull's-eye lesion but rather an uncharacteristic variant as reported in 25-30% of cases.4,5 This may require referral to a specialist or prompt further diagnostic testing, thereby delaying the start of antibiotic therapy at the time for the best chance of cure without sequelae.

[0004] These clinical hurdles to diagnosis are compounded by the limitations of current laboratory tests. The most commonly used assays are serologic. However, these assays have been hampered by the biologically delayed antibody response (often 3 weeks or more to reach detectable levels). They are further limited because a single positive test is an indirect measure of exposure and cannot distinguish active from past infection. A direct Borrelia molecular test such as PCR can have high specificity but typically the tests have lacked sensitivity when applied to blood. In the past, PCR assays for Lyme disease did not have the benefit of current advances in sample preparation.6 and enhancement methods.7,8 such as Borrelia-targeted DNA enrichment (akin to a "molecular culture") that may allow detection of a low number of nucleic acid copies in the blood. In addition, the optimal blood volume and component of the sample, such as whole blood versus serum, may have contributed to prior limitations of PCR based diagnostics.

SUMMARY OF THE INVENTION



[0005] The present invention provides methods of detecting Lyme disease comprising whole blood nucleic acid extraction methods, as well as nested isothermal amplification methods. In certain embodiments, these methods are applied to detecting Lyme disease in patients without classic erythema migrans skin lesions.

[0006] In particular, the present invention provides a method of detecting Lyme disease, comprising: (1) a method of nucleic acid extraction comprising: (a) contacting a sample of whole blood with beads, a proteinase, and an anionic surfactant to generate a treated sample; (b) homogenizing said treated sample to generate a cell lysate; (c) centrifuging said cell lysate comprising a supernatant; (d) separating said supernatant from said cell lysate; (e) adding magnetic particles and lysis buffer to said supernatant to generate a magnetic-particle sample, wherein said magnetic particles are configured to bind nucleic acid molecules; (f) washing said magnetic-particle sample with a wash buffer; (g) treating said magnetic-particle sample in order to generate a dried magnetic bead sample; and (h) treating said dried magnetic bead sample with an elution buffer such that a purified nucleic acid sample is generated that comprises purified nucleic acid; and (2) a nested isothermal amplification method comprising: (a) contacting the purified nucleic acid sample with a buffer, dNTPs, and a plurality of nested PCR primer pairs configured to amplify at least part of at least one nucleic acid sequence from Borrelia burgdorferi; (b) incubating said purified nucleic acid sample with a DNA polymerase under isothermal conditions such that amplified nucleic acid is generated; (c) inactivating said DNA polymerase; and (d) subjecting said amplified nucleic acid to mass spectrometry to identify Borrelia burgdorferi in the sample.

[0007] In some embodiments, the mass spectrometry comprises electrospray ionization mass spectrometry and base composition analysis. In certain embodiments, the DNA polymerase comprises BstE DNA polymerase. In additional embodiments, the incubating is conducted at about 56 degrees Celsius. In further embodiments, inactivating the DNA polymerase comprises heating the sample to at least about 80 degrees Celsius. In other embodiments, the plurality of nested PCR primer pairs comprises at least 10 primer pairs (e.g., 10 ... 15 ... 19, etc). In particular embodiments, the plurality of nested PCR primer pairs comprises at least 20 primer pairs (e.g., 20 ... 25 ... 35 ... 45 ... etc.). In further embodiments, the at least one nucleic acid sequence from Borrelia burgdorferi comprises at least five nucleic acid sequences from Borrelia burgdorferi. In certain embodiments, the methods further comprise a step after (2)(c) but before (2)(d) of further amplifying the amplified nucleic acid without any purification of the amplified nucleic acid.

DESCRIPTION OF THE FIGURES



[0008] Figure 1 shows an atypical EM lesion from a patient who was PCR positive and seronegative with a negative ELISA after eight days of illness. Repeat serology at the end of therapy 3 weeks later showed a positive ELISA, positive IgM western blot and negative IgG western blot.

DETAILED DESCRIPTION



[0009] The present invention provides methods of detecting Lyme disease comprising whole blood nucleic acid extraction methods, as well as nested isothermal amplification methods. In certain embodiments, these methods are applied to detecting Lyme disease in patients without classic erythema migrans skin lesions.

[0010] The methods, kits, and compositions disclosed herein are useful with any target nucleic acid sequence and are not limited to any particular target sequence (e.g., not limited to nucleic acid sequences from B. burgdorferi). The discussion below is focused on detecting sequence from B. burgdorferi. Such target sequences are exemplary and are not intended to limit the scope of the present description. Other target sequences (e.g., from infections disease) are well known in the art. Also well know in the art are primers, including nested prior sets, that are useful in the present description.

[0011] Lyme disease is representative of an infectious disease where early diagnosis is imperative to avoid sequelae. However diagnosis is often difficult because the clinical manifestations, including the rash, are variable and the pathogens are often not in abundance, not easily cultured, and the antibody response to them is delayed. In the past PCR for B. burgdorferi in blood had low sensitivity. This may be related to low copy number, insufficient volume of blood or targeting the wrong component of blood

[0012] The present invention overcomes some of these obstacles by combining a pre-PCR nucleic acid enrichment with sensitive PCR detection from nucleic acid extraction from 1.25 ml of whole blood from patients with skin lesions and early Lyme disease. With this strategy, PCR positivity was found in 14 of 29 (p=0·0001) endemic area subjects with typical erythema migrans (EM) or a variant, often before seroconversion was positive. The enrichment technique increased the yield from 2 to 14. None of 44 control subjects were positive. A serendipitous and unexpected finding of clinical importance was the observation that 8 of 14 (57%) of PCR positive subjects had an atypical EM.

[0013] The invention provides improved early diagnosis of Lyme disease by combination of a pre-PCR Borrelia DNA enhancement, sensitive PCR, and targeting sufficient volumes of whole blood. The surprise finding of non-classic EM lesions in the majority of microbiologically proven Lyme disease cases serves as alert to clinicians evaluating patients with endemic area exposure.

[0014] In certain embodiments, the PCR generated amplicons are detected by mass spectrometry methods using bioagent identifying amplicons. In some embodiments, primers are selected to hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which flank variable sequence regions to yield a bioagent identifying amplicon which can be amplified and which is amenable to base composition analysis. In some embodiments, the corresponding base composition of one or more different amplicons is queried against a database of base compositions indexed to bioagents and to the primer pair used to generate the amplicon. A match of the measured base composition to a database entry base composition associates the sample bioagent to an indexed bioagent in the database. Thus, the identity of the unknown bioagent is determined. No prior knowledge of the unknown bioagent is necessary to make an identification. In some instances, the measured base composition associates with more than one database entry base composition. Thus, a second/subsequent primer pair is generally used to generate an amplicon, and its measured base composition is similarly compared to the database to determine its identity in triangulation identification. Furthermore, the methods of the invention can be applied to rapid parallel multiplex analyses, the results of which can be employed in a triangulation identification strategy. Thus, in some embodiments, the present invention provides rapid throughput and does not require nucleic acid sequencing or knowledge of the linear sequences of nucleobases of the amplified target sequence for bioagent detection and identification.

[0015] Methods of employing base compositions, databases containing base composition entries, and triangulation using primers, are described in the following patents, patent applications and scientific publications: US patent numbers 7,108,974; 7,217,510; 7,226,739; 7,255,992; 7,312,036; 7,339,051; US patent publication numbers 2003/0027135; 2003/0167133; 2003/0167134; 2003/0175695; 2003/0175696; 2003/0175697; 2003/0187588; 2003/0187593; 2003/0190605; 2003/0225529; 2003/0228571; 2004/0110169; 2004/0117129; 2004/0121309; 2004/0121310; 2004/0121311; 2004/0121312; 2004/0121313; 2004/0121314; 2004/0121315; 2004/0121329; 2004/0121335; 2004/0121340; 2004/0122598; 2004/0122857; 2004/0161770; 2004/0185438; 2004/0202997; 2004/0209260; 2004/0219517; 2004/0253583; 2004/0253619; 2005/0027459; 2005/0123952; 2005/0130196 2005/0142581; 2005/0164215; 2005/0266397; 2005/0270191; 2006/0014154; 2006/0121520; 2006/0205040; 2006/0240412; 2006/0259249; 2006/0275749; 2006/0275788; 2007/0087336; 2007/0087337; 2007/0087338 2007/0087339; 2007/0087340; 2007/0087341; 2007/0184434; 2007/0218467; 2007/0218467; 2007/0218489; 2007/0224614; 2007/0238116; 2007/0243544; 2007/0248969; WO2002/070664; WO2003/001976; WO2003/100035; WO2004/009849; WO2004/052175; WO2004/053076; WO2004/053141; WO2004/053164; WO2004/060278; WO2004/093644; WO 2004/101809; WO2004/111187; WO2005/023083; WO2005/023986; WO2005/024046; WO2005/033271; WO2005/036369; WO2005/086634; WO2005/089128; WO2005/091971; WO2005/092059; WO2005/094421; WO2005/098047; WO2005/116263; WO2005/117270; WO2006/019784; WO2006/034294; WO2006/071241; WO2006/094238; WO2006/116127; WO2006/135400; WO2007/014045; WO2007/047778; WO2007/086904; WO2007/100397; and WO2007/118222.

[0016] Exemplary base-count related methods and other aspects of use in the methods of the invention are also described in, for example, Ecker et al., Ibis T5000: a universal biosensor approach for microbiology. Nat Rev Microbiol. 2008 Jun 3.; Ecker et al., The Microbial Rosetta Stone Database: A compilation of global and emerging infectious microorganisms and bioterrorist threat agents.; Ecker et al., The Ibis T5000 Universal Biosensor: An Automated Platform for Pathogen Identification and Strain Typing.; Ecker et al., The Microbial Rosetta Stone Database: A common structure for microbial biosecurity threat agents.; Ecker et al., Identification of Acinetobacter species and genotyping of Acinetobacter baumannii by multilocus PCR and mass spectrometry. J Clin Microbiol. 2006 Aug;44(8):2921-32.; Ecker et al., Rapid identification and strain-typing of respiratory pathogens for epidemic surveillance. Proc Natl Acad Sci U S A. 2005 May 31;102(22):8012-7. Epub 2005 May 23.; Wortmann et al., Genotypic evolution of Acinetobacter baumannii Strains in an outbreak associated with war trauma, Infect Control Hosp Epidemiol. 2008 Jun;29(6):553-555.; Hannis et al., High-resolution genotyping of Campylobacter species by use of PCR and high-throughput mass spectrometry. J Clin Microbiol. 2008 Apr;46(4): 1220-5.; Blyn et al., Rapid detection and molecular serotyping of adenovirus by use of PCR followed by electrospray ionization mass spectrometry. J Clin Microbiol. 2008 Feb;46(2):644-51.; Eshoo et al., Direct broad-range detection of alphaviruses in mosquito extracts, Virology. 2007 Nov 25;368(2):286-95.; Sampath et al., Global surveillance of emerging Influenza virus genotypes by mass spectrometry. PLoS ONE. 2007 May 30;2(5):e489.; Sampath et al., Rapid identification of emerging infectious agents using PCR and electrospray ionization mass spectrometry. Ann N Y Acad Sci. 2007 Apr;1102: 109-20.; Hujer et al., Analysis of antibiotic resistance genes in multidrug-resistant Acinetobacter sp. isolates from military and civilian patients treated at the Walter Reed Army Medical Center. Antimicrob Agents Chemother. 2006 Dec;50(12):4114-23.; Hall et al., Base composition analysis of human mitochondrial DNA using electrospray ionization mass spectrometry: a novel tool for the identification and differentiation of humans. Anal Biochem. 2005 Sep 1;344(1):53-69.; Sampath et al., Rapid identification of emerging pathogens: coronavirus. Emerg Infect Dis. 2005 Mar;11(3):373-9.; Jiang Y, Hofstadler SA. A highly efficient and automated method of purifying and desalting PCR products for analysis by electrospray ionization mass spectrometry; Jiang et al., Mitochondrial DNA mutation detection by electrospray mass spectrometry; Russell et al., Transmission dynamics and prospective environmental sampling of adenovirus in a military recruit setting; Hofstadler et al., Detection of microbial agents using broad-range PCR with detection by mass spectrometry: The TIGER concept. Chapter in; Hofstadler et al., Selective ion filtering by digital thresholding: A method to unwind complex ESI-mass spectra and eliminate signals from low molecular weight chemical noise.; Hofstadler et al., TIGER: The Universal Biosensor.; Van Ert et al., Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis.; Sampath et al., Forum on Microbial Threats: Learning from SARS: Preparing for the Next Disease Outbreak -- Workshop Summary (ed. Knobler SE, Mahmoud A, Lemon S.) The National Academies Press, Washington, D.C. 2004. 181-185.

EXAMPLES


EXAMPLE 1


Early Lyme patient specimens.



[0017] The current Example is part of a larger, ongoing longitudinal cohort study of early Lyme disease being conducted in a suburban community of a medium-sized, Northeast city since the summer of 2008. Adult patients with early, untreated Lyme disease are referred to a primary care physician with infectious disease training (JA) and provide written consent to participate.

[0018] Eligible patients are required to be treatment naïve, to have a documented rash diagnosed as EM at time of enrollment, and to have evidence of systemic infection; typically manifest as dissemination of the primary EM lesion or concurrent onset of new flu-like or other symptoms. Patients with a prior history of Lyme disease or symptom duration of their current illness of longer than 3 months are excluded. 20. Forty four negative control specimens were obtained from Biomed Supply Inc. (Carlsbad, CA). Specimens were collected at a donation site in Pennsylvania from healthy donors screened by Biomed Supply Inc. A paired 7mL tube of EDTA treated whole blood and 5-12mL of serum was provided for each control patient.

Serological and other analyses.



[0019] At the initial, pre-treatment study visit, self-report demographics, medical history information, complete blood counts (CBC), complete metabolic panels (CMP) and 2-tier antibody testing for B. burgdorferi were performed as part of the patient clinical evaluation. All serologic testing for both the patient specimens and the negative controls was performed through the same commercial laboratory, and results were interpreted according to CDC criteria 11. Based on these criteria, approximate illness duration was documented and included in determining serostatus; patients with less than 4 weeks illness duration were positive based on either IgM or IgG positivity on western blot analysis, whereas patients with greater than 4 weeks illness duration were required to be IgG positive. Photos of EM rashes were reviewed by a panel of experienced clinicians without knowledge of serologic or PCR status. Skin lesions with classic bull's eye appearance with central clearing and peripheral erythema were classified as classic EM lesions. EM with uniform red or red-blue appearance which lacked central clearing were classified as non-classic EM lesions.

DNA extraction from blood specimens:



[0020] A combination of bead-beating and magnetic bead isolation was used to extract nucleic acids from 1.25mL of whole EDTA blood. The blood was mixed in 2.0 mL screw-cap tubes (Sarstedt, Newton NC) filled with 1.35 g of 0.1-mm yttriastabilized zirconium oxide beads (Glen Mills, Clifton, NJ), 25 µL proteinase K solution (Qiagen, Valencia, CA), 142 µL of 20% SDS solution (Ambion, Austin, TX) and 1uL of DNA extraction control (Abbott Molecular, Des Plains, IL). The mixture was then homogenized in a Precellys 24 tissue homogenizer (Bioamerica Inc., Miami, FL) at 6,200 rpm for 3 sets of 90 sec with 5 sec between sets. The homogenized lysates were then incubated at 56°C for 15 min and then centrifuged for 3 min at 16,000 g in a bench top microcentrifuge. To isolate nucleic acids 1 mL of the supernatant was transferred to a 24-well deep-well Kingfisher plate (Thermo Scientific, Waltham, MA) along with 1.1 mL of Abbott lysis buffer (Abbott Molecular), and 160 µL of magnetic particles (Abbott Molecular). The specimens were incubated for 16.5 minutes in the lysis buffer at 56°C. Specimens were then washed once in Wash buffer I (Abbott Molecular), and three times in Wash buffer II (Abbott Molecular), with 1 min incubation for each wash step. The magnetic beads were then dried for 3 min at 65°C, and nucleic acids were eluted into 250 µL of elution buffer (Abbott Molecular), by incubating the magnetic particles at 65°C for 3 min.

Nested isothermal amplification of B. burgdorferi target DNA.



[0021] To increase sensitivity of B. burgdorferi detection by PCR/ESI-MS the seven target regions (eight primer pairs) of the previously described Borrelia genotyping assay21 were enriched by a nested isothermal amplification. For each of the seven target regions were amplified using 50 oligonucleotide primers: 25 upstream and 25 downstream to the DNA region of interest (Table 1).



















[0022] Since two of the target regions are close a single set of flanking oligos was designed to cover both targets. All primers were brought up to an initial concentration of 1mM in 10mM Tris pH 8.0 and 50uM EDTA (pH 8.0). The primers were mixed in equal proportions to create a 1mM oligo mix. Primers were designed using B. burgdorferi B31 genome sequence (gi|15594346) and have a GC content of 25 to 50%, spaced 6-10 nucleotides apart and have a target TM of 52°C. To ensure removal of any contaminating salts the pooled primers were dialyzed twice for 4 hours at 4°C against a 4L solution containing 10mM Tris pH 8.0 and 50uM EDTA pH 8.0 using a 5mL Float-A-Lyzer G2 (Spectrum Laboratories, Rancho Dominguez, CA) with a molecular weight cutoff of 0.5-1kDa.

[0023] The nested isothermal amplification was performed in a 225ul reaction in a 0.6mL PCR tube (Axygen Inc., Union City, California) containing 200uL of nucleic acid extract, 22.5ul Ibis 10X PCR Buffer II 21 (Ibis Biosciences, Carlsbad, CA), 0.2uM dNTPs (Bioline, Tauton, MA), and 10uM oligo mix. The reactions were incubated at 95°C for 10min followed by a cooling to 56°C in a MJ Thermocycler (Bio-Rad Laboratories, Hercules, CA.). The reactions were then removed to a heat block at 56°C and 11.25U of BstE DNA polymerase (Lucigen, Middleton, WI) enzyme added and the reactions incubated at 56°C for 2 hours followed by an 80°C heat inactivation for 20min. The resulting reaction was used directly in the subsequent PCR without purification.

Detection of B. burgdorferi from whole blood from patients with clinically diagnosed early Lyme disease.



[0024] Borrelia was detected by processing 2ul of Borrelia enriched nucleic acid extracts per PCR reaction on a previously described broad-range PCR/ESI-MS assay designed to detect and characterize Borrelia burgdorferi as previously described. 21. Electrospray ionization mass spectrometry was performed on the PLEX-ID biosensor (Abbott Molecular). Briefly, after PCR amplification, 30 µL aliquots of each PCR reaction were desalted and analyzed by mass spectrometry as previously described 21-23. Analysis of amplicons from any one of the eight primer pairs in the assay can be used to positively identify Borrelia DNA in a specimen.

RESULTS



[0025] The data are supportive that this new strategy can positively impact current clinical and laboratory impediments to early diagnosis of Lyme disease. A serendipitous finding of clinical importance was that the majority (∼60%), of rashes were not the classic EM in PCR positive cases.

Detection of Lyme disease in 29 patients with classic and non-classic EM



[0026] Examination of nucleic acid extract from 1.25 ml of whole blood from the independent set of 29 endemic patients with clinically diagnosed Lyme disease with classic or non-classic EM and 44 healthy controls showed that 14 of the 29 Lyme patients, and 0 of 45 controls, (48%, p=0.0001) had detectable B. burgdorferi. The pre-PCR enrichment step enabled increased detection of Borrelia from 2 to 14 cases (7X) when assessed by the PCR/ESI-MS assay. Two-tiered serology was positive in 14 of the 29 specimens, not all of which overlapped with the 14 specimens positive by NIA/PCR/ESI-MS from Table 2. Nine of the clinically defined early Lyme disease specimens tested negative by both PCR and serology assays. However, we did not have skin isolates to determine if the Borrelia was actually present as a skin-restricted strain which did not disseminate to blood.23,24 Table 2 is presented to show PCR detection is possible even when serology is negative or not yet positive. The data support the concept that unambiguous diagnosis can be made early, before seroconversion.

Of the 14 early Lyme specimens that tested positive by the NIA/PCR/ESI-MS assay, 6 were negative by the 2-tiered test at the time of presentation before the initiation of antibiotics (Table 2). Of these 6 specimens, 4 seroconverted by the follow-up serological testing 3 weeks later indicating that these were likely recent infections. Two of the NIA/PCR/ESI-MS positive specimens (#48 and #51) did not seroconvert sufficiently to meet the CDC surveillance criteria25 but both specimens showed an increase in ELISA titers and increased IgG/IgM blot reactivity, indicating that these patients were most likely represent weak or slow responders possibly due to antibiotic therapy26 or for unknown reasons. Of the 44 control patient sera, a single sample was seropositive by ELISA and IgG western blot, but was PCR negative. This low rate in our sample set from the eastern United States is consistent with the known background seroreactivity from remote exposure in an endemic region.

[0027] These results demonstrate that B. burgdorferi infection can be diagnosed early, and even prior to seroconversion, from whole blood in a high percentage of patients with suspected Lyme using a pre-PCR Borrelia-DNA enhancing method coupled with PCR/ESI-MS assay. Other isothermal amplification methods to generate genetic material has been described but not for Borrelia.7,8 In this case, a novel nested isothermal amplification (NIA) was coupled with a PCR/ESI-MS assay and results were contrasted with the two-tier serology. However, in a clinical situation, finding Borrelia DNA circulating in blood in an ill person with endemic area exposure will have such a strong possibility of a current infection that it would support a clinical decision to treat regardless of whether there is a classical EM, an atypical skin lesion, or extracutaneous systemic symptoms and signs suggestive of Lyme disease. The could allow for early diagnosis of the 20-30% of patients with early Lyme disease that present with "viral-like" symptoms indistinguishable for other community acquired infectious diseases. In contrast, a single serologic test, even if positive, may be difficult to interpret in a person living in an endemic area because this single result could represent past exposure. The present strategy has the potential to remove this ambiguity.

[0028] Work conducted during development of embodiments of the present invention led to the conclusion that whole blood may be preferable to serum or plasma for detection of Borrelia. While not limited to any mechanism, this may be due to the pathogen adhering to particular blood cells. In contrast to many diagnostics based on analysis of small (ul) of samples, the present disclosure is configured to handle larger volumes (mls), but within clinical practice volumes, to increase the detection of pathogens that may be present only in low copy numbers. A recent paper27 showed that B. burgdorferi may be present in a high percentage of patients with Lyme disease but may require "teasing" it out with combinations of cultures and PCR. Although this method is not as easily performed within a clinically useful time period, as that which is described herein, it does suggest that there is an attainable target should it be present.

[0029] A serendipitous observation in this Example and an important clinical finding was the high percentage (57%) of PCR positive patients whose skin rash was not the classical EM but an atypical lesion (see Figure 1) that could be easily misdiagnosed. Photographs of lesions (all greater than 5 cm) were presented to four physicians experienced with Lyme disease, including two dermatologists. Without knowledge of the laboratory data they were asked to categorize the lesions into 1) those that they would expect any reasonably trained physician to recognize as EM and 2) into those that they would not fault such a physician from referring the patient or desiring supportive laboratory tests or more time for observation.28,29 Based on previous reports, including a vaccine trial, of atypical rashes in 25-30% of microbiologically confirmed B. burgdorferi infected subjects4,5 it was expected a similar percentage, but were surprised by almost 60%. This high percentage serves as an alert for physicians to consider Lyme disease in the differential diagnosis of patients with endemic area exposure and a rash that is not the classic EM. These atypical lesions serve as a reminder of the need for diagnostics for the endemic area patient who only has a flu-like illness during the high season for Lyme disease. Such a patient may benefit from the testing strategy described in this Example or, at the very least, closer monitoring.

[0030] It is believed that these results demonstrate a useful strategy to the diagnosis of infections when there is a paucity of pathogens or the immune response cannot provide an unambiguous result. It is likely that many of these difficult to diagnose infections will require a battery of diagnostic platforms each with advantages depending on when the patient presents.

REFERENCES



[0031] 

1 Benach JL, Bosler EM, Hanrahan JP et al. Spirochetes isolated from the blood of two patients with Lyme disease. N Engl J Med 1983;308:740-2.

2 Steere AC. Lyme disease. N Engl J Med 1989;321:586-96.

3 Steere AC. Lyme disease. N Engl J Med 2001;345:115-25.

4 Steere AC, Sikand VK, Meurice F et al. Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. Lyme Disease Vaccine Study Group. N Engl J Med 1998;339:209-15.

5 Berger BW, Johnson RC, Kodner C, Coleman L. Cultivation of Borrelia burgdorferi from erythema migrans lesions and perilesional skin. J Clin Microbiol 1992;30:359-61.

6 Crowder CD, Rounds MA, Phillipson CA et al. Extraction of total nucleic acids from ticks for the detection of bacterial and viral pathogens. J Med Entomol 2010;47:89-94.

7 Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 2009;15:62-9.

8 Notomi T, Okayama H, Masubuchi H et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28:E63.

9 Crowder CD, Matthews HE, Schutzer S et al. Genotypic variation and mixtures of Lyme Borrelia in Ixodes ticks from North America and Europe. PLoS ONE 2010;5:e10650.

10 Cox-Foster DL, Conlan S, Holmes EC et al. A metagenomic survey of microbes in honey bee colony collapse disorder. Science 2007;318:283-7.

11 Briese T, Palacios G, Kokoris M et al. Diagnostic system for rapid and sensitive differential detection of pathogens. Emerg Infect Dis 2005;11:310-3.

12 Eckburg PB, Bik EM, Bernstein CN et al. Diversity of the human intestinal microbial flora. Science 2005;308:1635-8.

13 Ecker DJ, Sampath R, Blyn LB et al. Rapid identification and strain-typing of respiratory pathogens for epidemic surveillance. Proc Natl Acad Sci U S A 2005;102:8012-7.

14 Hofstadler SA, Sampath R, Blyn LB et al. TIGER: the universal biosensor. Intl J Mass Spectrometry 2005;242:23-41.

15 Jacobs MR, Bajaksouzian S, Bonomo RA et al. Occurrence, distribution and origins of serotype 6C Streptococcus pneumoniae, a recently recognized serotype. J Clin Microbiol 2008.

16 Ecker DJ, Sampath R, Massire C et al. Ibis T5000: a universal biosensor approach for microbiology. Nat Rev Microbiol 2008;6:553-8.

17 Hannis JC, Manalili SM, Hall TA et al. High-resolution genotyping of Campylobacter species by use of PCR and high-throughput mass spectrometry. J Clin Microbiol 2008;46:1220-5.

18 Blyn LB, Hall TA, Libby B et al. Rapid detection and molecular serotyping of adenovirus by use of PCR followed by electrospray ionization mass spectrometry. J Clin Microbiol 2008;46:644-51.

19 Eshoo MW, Whitehouse CA, Zoll ST et al. Direct broad-range detection of alphaviruses in mosquito extracts. Virology 2007;368:286-95.

20 Sampath R, Russell KL, Massire C et al. Global surveillance of emerging Influenza virus genotypes by mass spectrometry. PLoS ONE 2007;2:e489.

21 Sampath R, Hall TA, Massire C et al. Rapid identification of emerging infectious agents using PCR and electrospray ionization mass spectrometry. Ann N Y Acad Sci 2007;1102:109-20.

22 Sampath R, Hofstadler SA, Blyn LB et al. Rapid identification of emerging pathogens: coronavirus. Emerg Infect Dis 2005;11:373-9.

23 Seinost G, Dykhuizen DE, Dattwyler RJ et al. Four clones of Borrelia burgdorferi sensu stricto cause invasive infection in humans. Infect Immun 1999;67:3518-24.

24 Wormser GP, Liveris D, Nowakowski J et al. Association of specific subtypes of Borrelia burgdorferi with hematogenous dissemination in early Lyme disease. J Infect Dis 1999;180:720-5.

25 CDC. Recommendations for test performance and interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease. MMWR Morb Mortal Wkly Rep 1995;44:590-1.

26 Dattwyler RJ, Volkman DJ, Luft BJ, Halperin JJ, Thomas J, Golightly MG. Seronegative Lyme disease. Dissociation of specific T- and B- lymphocyte responses to Borrelia burgdorferi. N Engl J Med 1988;319:1441-6.

27 Liveris D, Schwartz I, Bittker S et al. Improving the Yield of Blood Cultures in Early Lyme Disease. Journal of Clinical Microbiology 2011;JCM.

28 Berger BW. Erythema migrans. Clin Dermatol 1993;11:359-62.

29 Berger BW. Dermatologic manifestations of Lyme disease. Rev Infect Dis 1989;11 Suppl 6:S1475-S1481.



[0032] Various modification and variation of the described methods of the invention will be apparent to those skilled in the art. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

SEQUENCE LISTING



[0033] 

<110> IBIS BIOSCIENCES, INC.

<120> SAMPLE PREPARATION METHODS

<130> 11211WOO1

<140> PCT/US2012/053909
<141> 2012-09-06

<150> 61/531,471
<151> 2011-09-06

<160> 350

<170> PatentIn version 3.5

<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 1
ccgaaaaaga tgggctttt   19

<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 2
aggttaaaaa gtccgaaact att   23

<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 3
tctcccgatc aaattagaaa ttg   23

<210> 4
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 4
aaagagataa aagattttga aagaataaa   29

<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 5
aaagctaggt ttttggagtt tt   22

<210> 6
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 6
acagaaaaag aagaagaatt gattaa   26

<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 7
atgatgctgg gaatcaggtt c   21

<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 8
gggcttggac ttgatttg   18

<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 9
gtcttttaat gtgctaatgc aaga   24

<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 10
gcgttcctac taatgtatca ggg   23

<210> 11
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 11
ggcagagtta aaatatatga aaatatag   28

<210> 12
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 12
cacccttcaa gaacttttaa cag   23

<210> 13
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 13
ggctcttgaa gcttatgg   18

<210> 14
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 14
agacttggag aaatggagg   19

<210> 15
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 15
gaggaaaggc tcaatttgg   19

<210> 16
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 16
tcttgtttct cagcaacct   19

<210> 17
<211> 16
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 17
cgcaagatca acaggc   16

<210> 18
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 18
actacaccat cttgttgatg ata   23

<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 19
gtaatggttg gggtgattta c   21

<210> 20
<211> 16
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 20
ggagagccgt tcgaaa   16

<210> 21
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 21
ccaacttcta aagaaatttt atatgatgg   29

<210> 22
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 22
aggaaaaatt aaaaactgct gga   23

<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 23
tgtttttgaa tctgctacaa atga   24

<210> 24
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 24
ctggtaaata tcttggtgaa tcttataa   28

<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 25
ggacagttaa tggaatctca at   22

<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 26
ataccaaata tgagcaactg gggc   24

<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 27
aagcccaatc ctagagggta   20

<210> 28
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 28
tagaattcaa actagatgct gtaat   25

<210> 29
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 29
cggttcaatt actacatatt tttcata   27

<210> 30
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 30
gcccggttca attactaca   19

<210> 31
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 31
tcttcattta aaagctgcat tttt   24

<210> 32
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 32
gctctctagc ttctatgtac tca   23

<210> 33
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 33
aagcattaaa agacatacca tatcgc   26

<210> 34
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 34
gaagagtttt aatagcctca gccc   24

<210> 35
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 35
gacgaaagct catcaagatc a   21

<210> 36
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 36
cagttttatc atctttatct atcatttgaa   30

<210> 37
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 37
aaattctcaa taatttcaag acgtctt   27

<210> 38
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 38
atccactctg gcttattgcc a   21

<210> 39
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 39
ggggaataac aggaagaac   19

<210> 40
<211> 15
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 40
gctgaaccat tggcc   15

<210> 41
<211> 15
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 41
atgttgcaaa gcgcc   15

<210> 42
<211> 35
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 42
cgattatttc tatttatgac tcttctataa agatc   35

<210> 43
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 43
gcattaagaa gaagcaactt tct   23

<210> 44
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 44
attctttttt cgtttctcac aataatc   27

<210> 45
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 45
gtcaaaaaga gagtctactg attc   24

<210> 46
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 46
gaacctttga caacctttct ttta   24

<210> 47
<211> 15
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 47
cgacttgaga ggcct   15

<210> 48
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 48
cctgcttacc ttttaatgca t   21

<210> 49
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 49
ttttaccaag aagattttgc ctaa   24

<210> 50
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 50
caataacaga acgaccagaa taa   23

<210> 51
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 51
gtgttgctat gaatcctgtt g   21

<210> 52
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 52
ggtagaagac ccaaggt   17

<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 53
ggaaagctgg taaaagtagg   20

<210> 54
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 54
tggaaatgaa gattatgcca atattt   26

<210> 55
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 55
tttttaaaaa atgtattgca acaattgg   28

<210> 56
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 56
ttatcatctg gcgagatgag   20

<210> 57
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 57
gacgggaatt atgtcactg   19

<210> 58
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 58
ggtggatatg ctatgatact tg   22

<210> 59
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 59
gggtggacag cttataaga   19

<210> 60
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 60
cgttcacaat attgagctta atgt   24

<210> 61
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 61
cttacctctt gaaaatattc ctattgg   27

<210> 62
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 62
ctaatgctcc aattaaaatt ggc   23

<210> 63
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 63
ggttggagat gttttggaaa g   21

<210> 64
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 64
aaaggtatat tatttctcct aaaggc   26

<210> 65
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 65
gctaatatag ctttgcttgt ttataaag   28

<210> 66
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 66
gttgcttcta ttgaatatga tcctaa   26

<210> 67
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 67
cgaagagata aatttagcat tcctg   25

<210> 68
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 68
gcataagaga aagtataggt tgattg   26

<210> 69
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 69
ctggtaggat tagtattaga agaagag   27

<210> 70
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 70
aaaaggtaaa aaatttaaat cgggc   25

<210> 71
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 71
caaaggtaat gatcctttga aatc   24

<210> 72
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 72
gctataagac gactttatct tttgat   26

<210> 73
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 73
agacttataa gccaaaaact tcttc   25

<210> 74
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 74
ggttttggag aaaaataaat atggg   25

<210> 75
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 75
caaaaaggaa gataaaatag atatttttta gtg   33

<210> 76
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 76
tttcttgcga gtcttataac ct   22

<210> 77
<211> 38
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 77
tttatttctt cttttaataa taaatttatc tgaatatc   38

<210> 78
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 78
ttttttaata gatcttgcca ctatactc   28

<210> 79
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 79
actttttgat aaagactctt ttctataaaa g   31

<210> 80
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 80
cttctcactt ccaaaagacg   20

<210> 81
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 81
aagatctgga gtaggtttta ataac   25

<210> 82
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 82
ggcttaccat ttcaggaatt at   22

<210> 83
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 83
aagttttgcc attgtaaaca gatat   25

<210> 84
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 84
caagatcctc ggtaatataa ataggt   26

<210> 85
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 85
ctcgccaagc ttatgtc   17

<210> 86
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 86
cctctaaaaa tccttgtagg tg   22

<210> 87
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 87
cctctaaaaa tccttgtagg tg   22

<210> 88
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 88
cttccctttt tatctgactt agc   23

<210> 89
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 89
atcttctatt taccaacata actacttac   29

<210> 90
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 90
agggtaaatt tttgcccttt g   21

<210> 91
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 91
ctattggcct aacttttttt gg   22

<210> 92
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 92
agactctccc cggatatt   18

<210> 93
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 93
aaagcactgc aatagcc   17

<210> 94
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 94
taaaagctta gctcctttat tagg   24

<210> 95
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 95
tgatgctgct gacttaacaa   20

<210> 96
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 96
ctcggaaaga tttttattgt gataca   26

<210> 97
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 97
catcaaccat aactgtttta acaaatatc   29

<210> 98
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 98
gccaaatctt tttacgacga c   21

<210> 99
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 99
tatcagctct acccctagc   19

<210> 100
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 100
cttcaacaaa aatatgacaa tttctattaa c   31

<210> 101
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 101
cctatccttc tgccaacg   18

<210> 102
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 102
ggaaaaaaga ttgtatatac ttgacatg   28

<210> 103
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 103
caaagtagaa gaagatccaa gtattc   26

<210> 104
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 104
ggcaagaatt ttgggataat aaca   24

<210> 105
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 105
tgtctaaata cgaattcata aaaattgaaa   30

<210> 106
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 106
gaataagttg ttatgagtta attttcaaga   30

<210> 107
<211> 34
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 107
taaataaaat taacaaaaat gcaatctaaa agaa   34

<210> 108
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 108
cacagcatca aaattgttag c   21

<210> 109
<211> 15
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 109
ccgtgctggt tcaag   15

<210> 110
<211> 14
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 110
gaggggctag tggg   14

<210> 111
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 111
ggaagtggta gacacgc   17

<210> 112
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 112
aaaaaatata atggttaata gtgctgtg   28

<210> 113
<211> 34
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 113
gtaattaaaa aaaataaaaa agttgacaaa aatt   34

<210> 114
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 114
cgctgtaata gcaacaacaa taata   25

<210> 115
<211> 38
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 115
aataatattt tcaaaaataa aaataattat atttgcaa   38

<210> 116
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 116
ctctaagctt caaactaggt ca   22

<210> 117
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 117
aactttgctc tcaatagttg ttt   23

<210> 118
<211> 32
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 118
tatgaaatct aatctattta ttgtttctga ct   32

<210> 119
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 119
gcaatattta tgtcagcagg aa   22

<210> 120
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 120
gaaggtttat accctttgga g   21

<210> 121
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 121
tggtcaatat ggggtattaa ctttat   26

<210> 122
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 122
caataacctg cttgacaaaa taaatta   27

<210> 123
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 123
ggaaattaat gggaaataaa ttatttaaaa aca   33

<210> 124
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 124
agacataata tctttttaca ttgggaaa   28

<210> 125
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 125
tttaggaatt ttttggggag c   21

<210> 126
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 126
tgagggtgag ttcctgt   17

<210> 127
<211> 35
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 127
ttgtttttta aacttattaa tattttcttc tgtac   35

<210> 128
<211> 15
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 128
ggcaaacccc aaagc   15

<210> 129
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 129
gtgttctaat ttctcgatcc c   21

<210> 130
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 130
actgtgtcca tttatagtaa ttctc   25

<210> 131
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 131
ctagcccttt tttatataat tgcagg   26

<210> 132
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 132
gtaccataca ggcatttctt tta   23

<210> 133
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 133
ctagtaccgt tccaagct   18

<210> 134
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 134
gtccatctga agtttgaata atctc   25

<210> 135
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 135
acgctgtaag atcctctc   18

<210> 136
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 136
gtaattttta aaacccactg tcttaaata   29

<210> 137
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 137
cgtctagcaa tctttcagc   19

<210> 138
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 138
agattcaggc cattctaatt ct   22

<210> 139
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 139
tccaatttcg ctgcatttc   19

<210> 140
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 140
aattcaattt caactcctgt tgat   24

<210> 141
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 141
ttttatcgct gtggcctt   18

<210> 142
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 142
ctttacaaca aggccagac   19

<210> 143
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 143
cgatcactaa atatgtgatg cc   22

<210> 144
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 144
gttgttcttt gttatttttt ctattagttt att   33

<210> 145
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 145
cttcgtgctt tacatatttt aaaacatt   28

<210> 146
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 146
gagaagtcct attaagatcg ct   22

<210> 147
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 147
ctgtgaaaac tcccgattta tc   22

<210> 148
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 148
catttgttat tggatgaaat gcg   23

<210> 149
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 149
gcttccaacc caaattg   17

<210> 150
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 150
cggttccgta agttcct   17

<210> 151
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 151
tctgcttctc aaaatgtaag aaca   24

<210> 152
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 152
taaccaaatg cacatgttat caaa   24

<210> 153
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 153
ttgctgatca agctcaatat   20

<210> 154
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 154
gcaacttaca gacgaaatta at   22

<210> 155
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 155
agacagaggt tctatacaaa ttga   24

<210> 156
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 156
aggtaacggc acatattcag a   21

<210> 157
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 157
taagaatgaa ggaattggca gtt   23

<210> 158
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 158
aatttaaatg aagtagaaaa agtcttagt   29

<210> 159
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 159
ggctattaat tttattcaga caacaga   27

<210> 160
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 160
ttgtcacaag cttctagaaa ta   22

<210> 161
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 161
tttctggtaa gattaatgct caaat   25

<210> 162
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 162
gagcttctga tgatgctgct   20

<210> 163
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 163
gaaaagcttt ctagtgggta c   21

<210> 164
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 164
cattaacgct gctaatctta gtaa   24

<210> 165
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 165
catcagctat taatgcttca aga   23

<210> 166
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 166
catggaggaa tgatatatga ttatcatg   28

<210> 167
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 167
ttttttttta atttttgtgc tattcttttt aac   33

<210> 168
<211> 35
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 168
taataataat tatttttaat gctattgcta tttgc   35

<210> 169
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 169
attaaaggct tttgatttta atcaaaga   28

<210> 170
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 170
ttaagcgcat gaaagatcaa g   21

<210> 171
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 171
gtggaaggtg aacttaatac c   21

<210> 172
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 172
gattataaaa agaagtacga agatagagag   30

<210> 173
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 173
ttattttttt gattaaaaat tttcaagtcg taa   33

<210> 174
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 174
gcttccggag gagttattta t   21

<210> 175
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 175
taggagattg tctgtcgc   18

<210> 176
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 176
gcaacattag ctgcataaat at   22

<210> 177
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 177
tccctcacca gagaaaag   18

<210> 178
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 178
acaccctctt gaaccggtg   19

<210> 179
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 179
tgagaaggtg ctgtagcagg   20

<210> 180
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 180
ttgtaacatt aacaggagaa ttaactc   27

<210> 181
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 181
ttagcaagtg atgtattagc atca   24

<210> 182
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 182
tgatcactta tcattctaat agcattt   27

<210> 183
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 183
ctattttgga aagcacctaa at   22

<210> 184
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 184
gcatactcag tactattctt tatagat   27

<210> 185
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 185
tgagcataag atgcttttag attt   24

<210> 186
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 186
tctgtcattg tagcatcttt ta   22

<210> 187
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 187
ttaaaatact attagttgtt gctgctac   28

<210> 188
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 188
attagcctgc gcaatcat   18

<210> 189
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 189
gcaatgacaa aacatattgg g   21

<210> 190
<211> 34
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 190
ttaatacaat ttataccaat taaactagaa tttt   34

<210> 191
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 191
ataaaaaaac aaaagatcct ttaaaggatc   30

<210> 192
<211> 35
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 192
ataaattata ctaaaattat taaatttttg ccgat   35

<210> 193
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 193
gcctgcatta tgctttataa ca   22

<210> 194
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 194
cctactcaaa gcaaactcc   19

<210> 195
<211> 34
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 195
cgaaaatact ttataacaat ctttaatttt aaca   34

<210> 196
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 196
tcgacttatc tgctttttgt taac   24

<210> 197
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 197
ctatctttgc catcttcata gtc   23

<210> 198
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 198
gcaataaaaa tagaagattc tttgtagat   29

<210> 199
<211> 34
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 199
taaaatttca ttttcataaa catcaagatt aata   34

<210> 200
<211> 14
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 200
gcccgacata ccca   14

<210> 201
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 201
ttaatgaaaa agaatacatt aagtgcaata t   31

<210> 202
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 202
ctaataattc ataaataaaa aggaggcac   29

<210> 203
<211> 32
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 203
ttttcaaata aaaaattgaa aaacaaaatt gt   32

<210> 204
<211> 34
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 204
aatatttatt caagatattg aagaatttga aaaa   34

<210> 205
<211> 35
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 205
tttaaaatca aattaagaca atatttttca aattc   35

<210> 206
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 206
agcatatttg gctttgctta tg   22

<210> 207
<211> 38
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 207
aaattaaaac tttttttatt aaagtatact tcatttaa   38

<210> 208
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 208
gcctgagtat tcattatata agtcc   25

<210> 209
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 209
tatattggga tccaaaatct aatacaag   28

<210> 210
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 210
caatttctct aattcttctt gcaattag   28

<210> 211
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 211
ggagtatagt aaggtattac ttttgtataa a   31

<210> 212
<211> 32
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 212
ttcctgagat attcatattt ttaatttctt tt   32

<210> 213
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 213
gcaggacttc cacttagta   19

<210> 214
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 214
ggtaggagct tcttttgaat aaac   24

<210> 215
<211> 34
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 215
caaaataggt attttcaaat taaaaatttc cata   34

<210> 216
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 216
aatttaacaa ttatttgcat tccataacat a   31

<210> 217
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 217
gcttagagtc tttagatact aggc   24

<210> 218
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 218
aaagatttca gagctcccat at   22

<210> 219
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 219
ttctgaaaat aaaagagatt tttcatctc   29

<210> 220
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 220
tgactcatga taatttgaaa tttgtttg   28

<210> 221
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 221
aaattatcag gaattttttc aatactgtc   29

<210> 222
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 222
gcaatacaat tttttgtaaa agctaattg   29

<210> 223
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 223
ccgtaaattt tttgagtttc atttgat   27

<210> 224
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 224
agttacttct ggatggaatt gt   22

<210> 225
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 225
aatttttaat tatttgatca ccaaattcag   30

<210> 226
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 226
accgcattag aatccgtaat   20

<210> 227
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 227
acctctttca cagcaagtt   19

<210> 228
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 228
catctataga tgacagcaac g   21

<210> 229
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 229
ttaccaatag ctttagcagc a   21

<210> 230
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 230
gtatccaaac cattattttg gtgta   25

<210> 231
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 231
gctaacaatg atccattgtg attat   25

<210> 232
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 232
tattagggtt gatattgcat aagc   24

<210> 233
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 233
cttcattttt caatccatct aatttttg   28

<210> 234
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 234
ttagccgcat caattttttc c   21

<210> 235
<211> 32
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 235
tcttttaatt tattagtaaa tgtttcagaa ca   32

<210> 236
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 236
cttctttacc aagatctgtg tg   22

<210> 237
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 237
cttttgcatc agcatcagt   19

<210> 238
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 238
tttagtttta gtaccatttg tttttaaaat g   31

<210> 239
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 239
gattcaaata attttccaag ttcttcag   28

<210> 240
<211> 19
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 240
ctgcttttga caagacctc   19

<210> 241
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 241
tttaactgaa ttagcaagca tctc   24

<210> 242
<211> 15
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 242
ccacaacagg gcttg   15

<210> 243
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 243
gatcttaatt aaggtttttt tggactt   27

<210> 244
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 244
ccagttactt ttttaaaaca aattaatctt ata   33

<210> 245
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 245
agaaatcttt cttgacttat attgacttt   29

<210> 246
<211> 39
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 246
gaattttaag aaattttttg agaaaataaa aaaataaaa   39

<210> 247
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 247
tattctttaa gagaagagct taaagtt   27

<210> 248
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 248
aaattcaatt tattaacggc ttttgtaata   30

<210> 249
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 249
tctagcaccc aattttgttt atattta   27

<210> 250
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 250
gtttaagcct acttaaagtc tttaaaatc   29

<210> 251
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 251
cccacactct ctctttcaaa   20

<210> 252
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 252
gatattaacc ggcatttaac ctt   23

<210> 253
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 253
tctagcttac aatcccattt ataaga   26

<210> 254
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 254
ccttcaaatt ttaattttcc tctaaaagtt a   31

<210> 255
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 255
ccttcaaaag aagaatcaag atacaa   26

<210> 256
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 256
cacaccccct tttgaagata   20

<210> 257
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 257
gtaataacct tactattctt gccaata   27

<210> 258
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 258
ttctactatt aatgtatcac aaattaccac   30

<210> 259
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 259
gcatttacat tgcccttcaa   20

<210> 260
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 260
caaccgctgt ttaaataaac ctt   23

<210> 261
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 261
aatatttttt ttgtttttac atccccatat   30

<210> 262
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 262
cacacttacc atcaaaaatt atattatcat   30

<210> 263
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 263
aagaaaataa atctacaatt tcattagact tta   33

<210> 264
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 264
caaagtatct tttatttgtg aaacgg   26

<210> 265
<211> 34
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 265
tctacttatt attaattaat aaaaaacact gacc   34

<210> 266
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 266
ctctacgaat taaattttta agaaaggatt tta   33

<210> 267
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 267
atcaaatcca ccattttttt tatcca   26

<210> 268
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 268
ccaaccgcct tatttcac   18

<210> 269
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 269
ttttcaaatt atcttcaatc ttaaactctt tag   33

<210> 270
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 270
ttttagcaac aactttaacc acttt   25

<210> 271
<211> 17
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 271
tgtcacgcta gatgcag   17

<210> 272
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 272
ctttacgcca cttaaatctg c   21

<210> 273
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 273
aatcagaaaa tattaccccg tttg   24

<210> 274
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 274
atattatttt ctaaacctga agaaggaata t   31

<210> 275
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 275
cattaaaaaa tttgatgata ttactttgct c   31

<210> 276
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 276
gttttgctgt taaagtaagg aaattag   27

<210> 277
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 277
gctgctagaa aaaaatctcg tt   22

<210> 278
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 278
ctgctagaaa gcgaataatt cataa   25

<210> 279
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 279
gaatttttta aatttgttgc aaaaaaacta g   31

<210> 280
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 280
gcgggtaaga aagacgaa   18

<210> 281
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 281
gaaaaacgct gtatcaacat ga   22

<210> 282
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 282
attagaaatg taagtgtaaa aagtgaatta aaa   33

<210> 283
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 283
cgctctcgtc aaaatttaaa aag   23

<210> 284
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 284
cttgagaaaa aatgcatctg c   21

<210> 285
<211> 38
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 285
gatatattaa agctattgtt taataatatt attaagga   38

<210> 286
<211> 34
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 286
attaacttaa atctttgatt gactatattt gaat   34

<210> 287
<211> 32
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 287
aggtttttga atatattaat caaaactatt gt   32

<210> 288
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 288
attttgaata aaaaaatttc ttattccatg c   31

<210> 289
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 289
caaagaaaat catcagacaa aaaagg   26

<210> 290
<211> 35
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 290
gaatttgaat ttaacaataa aaattattta tgctt   35

<210> 291
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 291
gaattttttg aaaaaatttt tattgccag   29

<210> 292
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 292
ctgtgaaaga aaaattttta aaagtgaaat   30

<210> 293
<211> 32
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 293
caatatagtg ttattttatg agtttaggaa ag   32

<210> 294
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 294
ttttgttggg ggatttttca g   21

<210> 295
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 295
ttttgttggg ggatttttca g   21

<210> 296
<211> 42
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 296
gcaatatatt tattttttta tttatttgtt ttattgatat   42

<210> 297
<211> 34
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 297
gtattatgat tgctttattt gtttattaca tttc   34

<210> 298
<211> 36
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 298
gatattattt atcttgtact tatcttttta tgtttt   36

<210> 299
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 299
gtcccaaaat tggaaaattt tcc   23

<210> 300
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 300
tatttaaaga gcttaaaatt aagagaaaag atc   33

<210> 301
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 301
cgtgaagctg caagaaaa   18

<210> 302
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 302
tggaaaagca ataaaagctg ctg   23

<210> 303
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 303
tgttgtatat gaacatttat tggaaat   27

<210> 304
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 304
gcttggtaat tctgagataa gaaa   24

<210> 305
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 305
cctcaatttg aaggtcaaac aaa   23

<210> 306
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 306
attttaaaga ggggcttaca gct   23

<210> 307
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 307
gccatgaatg aagcttttaa a   21

<210> 308
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 308
ctcatgttat gggatttaga agtgg   25

<210> 309
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 309
ctgacaacat tctttctttt gttaa   25

<210> 310
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 310
tgttaatgtg gggcttaaat g   21

<210> 311
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 311
gcttttcaat cagaacctta tt 22

<210> 312
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 312
gagggtggga taaaatcttt tt   22

<210> 313
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 313
tggtaaagaa aaatcttcaa aattttat   28

<210> 314
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 314
cgataaaata tacatttcaa ttgaagataa   30

<210> 315
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 315
ggcttaaaga gcttgctttt   20

<210> 316
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 316
agattataat ttcgatgttc ttgaaaaa   28

<210> 317
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 317
cggattctga aatttttgaa acttt   25

<210> 318
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 318
ggggactaag gttacttttt t   21

<210> 319
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 319
agaagttgtg ggggaatctt ctgtt   25

<210> 320
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 320
ctttttcaaa aggtattccg actt   24

<210> 321
<211> 27
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 321
ggtttatgtt aatagagatg gaaaaat   27

<210> 322
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 322
ggttgtaaat gctctatctt cgtt   24

<210> 323
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 323
tggtaagttt aataaaggca cgtat   25

<210> 324
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 324
ccttgaactt gttttaacaa aattac   26

<210> 325
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 325
accgatattc atgaagagga g   21

<210> 326
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 326
tacccatttt agcacttcct cca   23

<210> 327
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 327
tggcaaaatg gcctgaaaaa   20

<210> 328
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 328
ttgttttctc aacattaagc atttt   25

<210> 329
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 329
atcattggtg ataaccttat cttct   25

<210> 330
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 330
actcctgcac caagagat   18

<210> 331
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 331
atcttgtgat aacgaagttt tgta   24

<210> 332
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 332
tccatcaaca tcggcatctg   20

<210> 333
<211> 24
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 333
aaaagctaaa agcaaagttc taat   24

<210> 334
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 334
atatatccat tttcaattaa atctctcat   29

<210> 335
<211> 20
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 335
tataaagagg aggcatggct   20

<210> 336
<211> 30
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 336
taaaaataat aaatacgatt gtcatacttt   30

<210> 337
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 337
tattgcgatt tttagtttca atagaa   26

<210> 338
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 338
cccaagccct ttatatctct gaa   23

<210> 339
<211> 18
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 339
agctgcgttg gattcatc   18

<210> 340
<211> 22
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 340
ctagcaggat ccatagttgt tt   22

<210> 341
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 341
atcatctata ttcatcaatc tcattttt   28

<210> 342
<211> 26
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 342
gagtaacaaa aattttttca gcttca   26

<210> 343
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 343
tttctgggct caactaaatc t   21

<210> 344
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 344
gattaattac attaagtgca ttctgttc   28

<210> 345
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 345
ccattaacgc tccaattaca c   21

<210> 346
<211> 31
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 346
tcctaacatt taatatttgt tctttatttt c   31

<210> 347
<211> 33
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 347
gcataattta aataagaagt ttttatttca tct   33

<210> 348
<211> 25
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 348
gaagagctct agaaacaata actga    25

<210> 349
<211> 23
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 349
tggtttaaga ccatctctta cgt   23

<210> 350
<211> 28
<212> DNA
<213> Artificial Sequence

<220>
<223> Description of Artificial Sequence: Synthetic primer

<400> 350
ctcatacata gaataaagta ttctcctg   28




Claims

1. A method of detecting Lyme disease, comprising:

(1) a method of nucleic acid extraction comprising:

a) contacting a sample of whole blood with beads, a proteinase, and an anionic surfactant to generate a treated sample;

b) homogenizing said treated sample to generate a cell lysate;

c) centrifuging said cell lysate comprising a supernatant;

d) separating said supernatant from said cell lysate;

e) adding magnetic particles and lysis buffer to said supernatant to generate a magnetic-particle sample, wherein said magnetic particles are configured to bind nucleic acid molecules;

f) washing said magnetic-particle sample with a wash buffer;

g) treating said magnetic-particle sample in order to generate a dried magnetic bead sample; and

h) treating said dried magnetic bead sample with an elution buffer such that a purified nucleic acid sample is generated that comprises purified nucleic acid; and

(2) a nested isothermal amplification method comprising:

a) contacting the purified nucleic acid sample with a buffer, dNTPs, and a plurality of nested PCR primer pairs configured to amplify at least part of at least one nucleic acid sequence from Borrelia burgdorferi;

b) incubating said purified nucleic acid sample with a DNA polymerase under isothermal conditions such that amplified nucleic acid is generated;

c) inactivating said DNA polymerase; and

d) subjecting said amplified nucleic acid to mass spectrometry to identify Borrelia burgdorferi in the sample.


 
2. The method of Claim 1, wherein said mass spectrometry bioagent analysis comprises electrospray ionization mass spectrometry and base composition analysis.
 
3. The method of Claim 1, wherein said DNA polymerase comprises BstE DNA polymerase.
 
4. The method of Claim 1, wherein said incubating is conducted at about 56 degrees Celsius.
 
5. The method of Claim 1, wherein said inactivating said DNA polymerase comprises heating said sample to at least about 80 degrees Celsius.
 
6. The method of Claim 1, wherein said plurality of nested PCR primer pairs comprises at least 10 primer pairs.
 
7. The method of Claim 1, wherein said plurality of nested PCR primer pairs comprises at least 20 primer pairs.
 
8. The method of Claim 1, wherein said at least one bioagent target sequence comprises at least five bioagent target sequences.
 
9. The method of Claim 1, further comprising a step after c) but before d) of further amplifying said amplified nucleic acid without any purification of said amplified nucleic acid.
 
10. The method of Claim 1, wherein the sample of whole blood is from a patient with skin lesions and early Lyme disease.
 
11. The method of Claim 1, wherein the sample of whole blood is from a patient without classic erythema migrans (EM) skin lesions.
 
12. The method of any preceding claim, wherein the sample of whole blood is 1.25mL of whole blood.
 


Ansprüche

1. Verfahren zum Nachweis von Lyme-Krankheit, das umfasst:

(1) ein Verfahren der Nukleinsäureextraktion, das umfasst:

a) in Kontakt bringen einer Vollblutprobe mit Perlen, einer Proteinase und einem anionischen oberflächenaktiven Mittel, um eine behandelte Probe zu erzeugen;

b) Homogenisieren der behandelten Probe, um ein Zelllysat zu erzeugen;

c) Zentrifugieren des Zelllysats, das einen Überstand umfasst;

d) Trennen des Überstands von dem Zelllysat;

e) Zugeben von Magnetpartikeln und Lysepuffer zu dem Überstand, um eine Magnetpartikelprobe zu erzeugen, wobei die Magnetpartikel so gestaltet sind, dass sie Nukleinsäuremoleküle binden;

f) Waschen der Magnetpartikelprobe mit einem Waschpuffer;

g) Behandeln der Magnetpartikelprobe, um eine getrocknete Magnetpartikelprobe zu erzeugen; und

h) Behandeln der getrockneten Magnetpartikelprobe mit einem Elutionspuffer, sodass eine gereinigte Nukleinsäureprobe erzeugt wird, die gereinigte Nukleinsäure umfasst; und

(2) ein ineinandergeschachteltes isothermes Amplifikationsverfahren, das umfasst:

a) in Kontakt bringen der gereinigten Nukleinsäureprobe mit einem Puffer, dNTPs und einer Vielzahl von ineinandergeschachtelten PCR-Primer-Paaren, die so gestaltet sind, dass sie mindestens einen Teil von mindestens einer Nukleinsäuresequenz von Borrelia burgdorferi amplifizieren;

b) Inkubieren der gereinigten Nukleinsäureprobe mit einer DNA-Polymerase unter isothermen Bedingungen, sodass amplifizierte Nukleinsäure erzeugt wird;

c) Inaktivieren der DNA-Polymerase; und

d) Durchführen einer Massenspektrometrie mit der amplifizierten Nukleinsäure, um Borrelia burgdorferi in der Probe zu identifizieren.


 
2. Verfahren nach Anspruch 1, wobei die Massenspektrometrie-Analyse des biologischen Agens Elektrospray-Ionisation-Massenspektrometrie und Grundzusammensetzungsanalyse umfasst.
 
3. Verfahren nach Anspruch 1, wobei die DNA-Polymerase BstE-DNA-Polymerase umfasst.
 
4. Verfahren nach Anspruch 1, wobei das Inkubieren bei ca. 56 °C durchgeführt wird.
 
5. Verfahren nach Anspruch 1, wobei das Inaktivieren der DNA-Polymerase das Erhitzen der Probe auf mindestens ca. 80 °C umfasst.
 
6. Verfahren nach Anspruch 1, wobei die Vielzahl der ineinandergeschachtelten PCR-Primer-Paare mindestens 10 Primer-Paare umfasst.
 
7. Verfahren nach Anspruch 1, wobei die Vielzahl der ineinandergeschachtelten PCR-Primer-Paare mindestens 20 Primer-Paare umfasst.
 
8. Verfahren nach Anspruch 1, wobei die mindestens eine Zielsequenz des biologischen Agens mindestens fünf Zielsequenzen des biologischen Agens umfasst.
 
9. Verfahren nach Anspruch 1, das weiter einen Schritt nach c) aber vor d) des weiteren Amplifizierens der amplifizierten Nukleinsäure umfasst, ohne jegliche Reinigung der amplifizierten Nukleinsäure.
 
10. Verfahren nach Anspruch 1, wobei die Vollblutprobe von einem Patienten mit Hautläsionen und früher Lyme-Krankheit stammt.
 
11. Verfahren nach Anspruch 1, wobei die Vollblutprobe von einem Patienten ohne klassische Erythema-migrans (EM)-Hautläsionen stammt.
 
12. Verfahren nach einem der vorhergehenden Ansprüche, wobei die Vollblutprobe 1,25 ml Vollblut ist.
 


Revendications

1. Procédé de détection de la maladie de Lyme, comprenant :

(1) un procédé d'extraction d'acide nucléique comprenant les étapes consistant à :

a) mettre en contact un échantillon de sang entier avec des billes, une protéinase et un surfactant anionique pour générer un échantillon traité ;

b) homogénéiser ledit échantillon traité pour générer un lysat de cellules ;

c) centrifuger ledit lysat de cellules comprenant un surnageant ;

d) séparer ledit surnageant à partir dudit lysat de cellules ;

e) ajouter des particules magnétiques et un tampon pour lyse au dit surnageant afin de générer un échantillon à particules magnétiques, dans lequel lesdites particules magnétiques sont configurées pour se lier à des molécules d'acide nucléique ;

f) laver ledit échantillon à particules magnétiques avec un tampon de lavage ;

g) traiter ledit échantillon à particules magnétiques dans le but de générer un échantillon séché à billes magnétiques ; et

h) traiter ledit échantillon séché à billes magnétiques avec un tampon d'élution, de sorte qu'un échantillon d'acide nucléique purifié soit généré, lequel comprend un acide nucléique purifié ; et

(2) un procédé d'amplification nichée isotherme comprenant les étapes consistant à :

a) mettre en contact l'échantillon d'acide nucléique purifié avec un tampon, des dNTP et une pluralité de paires d'amorces d'ACP nichée, configurées pour amplifier au moins une partie d'au moins une séquence d'acide nucléique provenant de Borrelia burgdorferi ;

b) incuber ledit échantillon d'acide nucléique purifié avec une ADN polymérase dans des conditions isothermes, de sorte qu'un acide nucléique amplifié soit généré ;

c) inactiver ladite ADN polymérase ; et

d) soumettre ledit acide nucléique amplifié à une spectrométrie de masse pour identifier Borrelia burgdorferi dans l'échantillon.


 
2. Procédé selon la revendication 1, dans laquelle ladite analyse de bio-agent par spectrométrie de masse comprend une spectrométrie de masse à ionisation par électro-pulvérisation et une analyse de la composition de base.
 
3. Procédé selon la revendication 1, dans laquelle ladite ADN polymérase comprend une ADN polymérase BstE.
 
4. Procédé selon la revendication 1, dans laquelle ladite incubation est conduite à environ 56 degrés Celsius.
 
5. Procédé selon la revendication 1, dans laquelle ladite inactivation de ladite ADN polymérase comprend un chauffage dudit échantillon jusqu'à au moins 80 degrés Celsius environ.
 
6. Procédé selon la revendication 1, dans laquelle ladite pluralité de paires d'amorces d'ACP nichée comprend au moins 10 paires d'amorces.
 
7. Procédé selon la revendication 1, dans laquelle ladite pluralité de paires d'amorces d'ACP nichée comprend au moins 20 paires d'amorces.
 
8. Procédé selon la revendication 1, dans laquelle ladite au moins une séquence cible de bio-agent comprend au moins cinq séquences cibles de bio-agent.
 
9. Procédé, selon la revendication 1, comprenant en outre une étape, après le paragraphe c) mais avant le d), d'amplification en outre dudit acide nucléique amplifié sans purification quelconque dudit acide nucléique amplifié.
 
10. Procédé selon la revendication 1, dans laquelle l'échantillon de sang entier provient d'un patient ayant des lésions cutanées et une maladie de Lyme précoce.
 
11. Procédé selon la revendication 1, dans laquelle l'échantillon de sang entier provient d'un patient dépourvu de lésions cutanées d'un érythème migrant (EM) classique.
 
12. Procédé selon l'une quelconque des revendications précédentes, dans laquelle l'échantillon de sang entier est de 1,25 mL de sang entier.
 




Drawing






REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description