TWI359196B - Primer pair, diagnosis kit and method for the dete - Google Patents

Description

1359196 > IX. Description of the Invention: [Technical Field 3 of the Invention] Field of the Invention The present invention relates to a primer pair for detecting a strain of 5 strains of sensu lato of Borrelia burgdorferi. It contains a forward primer designed according to the 16S rRNA gene of the bacterium and a reverse primer. The present invention also relates to a diagnostic kit 10 and method for detecting a pathogenic strain of Borrelia burgdorferi (5orre/za hMrgi/oz/en sensu lato) using the primer pair. L Prior Art 3 Background of the Invention Lyme disease is a global animal vector infectious disease, and its causative agent belongs to the genus Borrelia species 15 (7) 0 noisy.) Spirulina pathogenic strain sensu lato), and the pathogen is through a specific species of the genus Toxoplasma (/xoflfes sigh.) as a disease vector "pathogenic strain of Borrelia burgdorferi which has been pathogenic to humans in the past." (j^rre/ία wrgifor/erz. sensu lato) mostly occurs in the northern hemisphere with higher latitudes, such as one of the Borrelia burgdorferi strains 20, such as strains C®·such as rgdor/en·sensu stricto) North America and Western Europe 'B. burgdorferi (5· gan.m)·) and Borrelia genus (5. out) are more common in Europe, while Borrelia sinensis (and y卬 plus huayun are limited to In recent years, there have been many cases in Taiwan, where latitude is not high. Patients with Lyme disease usually have fever, sore throat, and fatigue in the early stages of infection.

/ 4 > 1359196 Tiredness, swollen lymph nodes, headache, chills and general fatigue. In disease vectors, such as tick, 'biting the human body for about 3 to 32 days (average about 7 to 10 days) (AC Steere ei a/, 86 (6): 685-98, 1977 Jan.), there is 60 to 80% of patients will have Erythema migrans (EM) at the site of bites 5 (D. Wright, Journal of the American Academy of Nurse Practitioners, 13 (5): 223-6; quiz 227 -8, 2001 May) » This migratory erythema gradually spreads with the proliferation of Borrelia, and then forms a migratory blush with a diameter greater than 5 cm. The migratory blush will appear red at the periphery and 10 in the middle. Whitening representation. After weeks or months, approximately 60% of patients will have a mid-term condition at the joint (E.M. Massarotti,

Clinics of North America, 86 (2): 297-309, 2002 March ; P.

Wang and E. Hilton, Frontiers in Bioscience, 6: B10-6, 2001 Sept. 1), nerve (AR Pachner, //wwwwo/ogica/ 183: 15 186-204, 2001 Oct.), heart (DS Pinto, 〇/

Dmen'ca" 86 (2): 285-96, Mar. 2002), Muscle (D. Wright, Journal of the American Academy of Nurse Practitioners, 13 (5): 223-6; quiz 227-8, May 2001) It is invaded by Borrelia. Among these conditions, the nervous system includes Bell's palsy, meningitis, and peripheral neuropathy. Aspects include heart rhythm block, pericarditis, myocarditis, etc. Late complications usually occur about 5 months after the onset of infection, mainly chronic arthritis, skin Chronic atrophic lesions, chronic meningitis 6 © 1359196 (chronic meningitis), peripheral neuritis 'back pain neuritis, numbness and even chronic fatigue (W. Burgdorfer. ei 〇/., Sciewce, 216:1317 -1319, 1982). If pregnant women are infected in the early or second phase of pregnancy, Borrelia will be transmitted to the fetus via the placenta, and 5 cause a deformed fetus or stillbirth (LE Markowitz with α/., 255:3394-3396 , 1986)

With regard to the detection of Lyme disease, serum antibody testing is currently the most commonly used clinical test method, mainly to detect specific antibodies produced in vivo after infection by patients with Borrelia. Commonly used serum antibody test methods include enzyme immunoassay (EIA) (P. Tugwell et al., Annals of Internal Medicine, 127 (12): 1109-23, 1997 Dec. 15; TB Ledue et al Journal of Clinical Microbiology, 34 (10): 2343-2350, 1996 Oct.) and Western blotting (SM Elizabath 15 et al., Journal of Clinical Microbiology, 33 (2): 419- 427, 1995 Feb. i F. Dressier et al., Journal of Infectious Diseases, 167:392-400,1993 ; Recommendations for Test Performance and interpretation from the second National Conference on Serologic Diagnosis of Lyme Disease. MMWR, August 11, 20 7995, #〇· 3/:5卯-597), and the applicable test samples for these two methods include blood and liquid samples and cerebrospinal fluid samples (GT. Stiernstedt ei α/., Journal of Clinical Microbiology, 21:819 -S25, 19 people 5). Enzyme immunoassay (K. Hansen ei α/. (1988), /ourmi/ 〇/ Clinical Microbiology, 26: 338-346; M. Karlsson et al. (1990),

7 1359196

Journal of Clinical Microbiology, 28..2M8-215Q) is mainly carried out on a microtiter plate, and the serum sample to be tested (after appropriate dilution treatment) is added to each well of the titration plate, and the purified Borsch is used. The flagella of Borrelia bacteria is used as an antigen tester 5, and forms an antigen-antibody immune complex (Ag-Ab immune complex) after binding to an antibody in a patient's serum sample. Thereafter, a conjugate having a horseradish peroxidase (HRP) and a substrate containing a chroman (substrate) are sequentially added to each well of the titration plate, and finally, The stop solution of sulfuric acid (H2S〇4) was stopped to stop the reaction. After the reaction is completed, the absorbance of the reaction mixture in each well of the titration plate is measured at a selected wavelength (e.g., 490 nm) using an enzyme immunoassay. Enzyme immunoassay uses a single gene strain (genospecies) as an antigen to test individual subtypes of Borrelia burgdorferi pathogenic strains (and .ftMrgi/or/eri 15 sensu lato), such as One of the Borrelia

Gene strain 〇β· ftwrgi/or/en· sensu. stricto), as for other subtypes, for example, B. burgdorferi (and, Borrelia oxysporum (5. α/ze) 舆 Japanese Borrelia ( 5./叩(10)ίαζ), there will also be cross-reactions. However, due to the limitation of antibody production time and individual immune response, the sensitivity of this 20 method is about 88%. Moreover, the flagellar antigen will have syphilis (syphilis), rheumatoid arthritis or antibodies produced in patients infected with other Borrelia bacteria have reacted and reacted, resulting in a false positive reaction (AR Mares ei β /., 〇 / M / Crotz.o/ogy, Nov. 2000, 38 (11): 4239-4241).

The operation process of the Western blot analysis method mainly includes: cultivating the pathogenic strain of Borrelia snail red body halogen (5. sensu lato) and then separating it, directly dissolving the separated bacteria, and brewing with polypropylene Polyacrylamide gel electrophoresis (PAGE) is used to separate protein components of different molecular weights. After electrophoresis, the separated protein band on the electrophoresis film was transferred to a nitrocellulose membrane (Nitrocellul〇se membrane) using an electric current disruptor (BioRad), and then the nitrocellulose was transferred. The membrane is cut into strips for clinical testing. If a sample to be tested contains a specific antibody against the pathogenic strain of Borrelia burgdorferi 〇Β· hrgifor/eW sensu lato), the antibody can bind to a protein located on the nitrocellulose membrane to form an antigen-antibody immunization Complex. Thereafter, a binding agent is added [eg, 'goat anti-human IgM/IgG conjugated with alkaline phosphatase'), and finally a substrate containing a coloring agent (BCIP/NBT) is added. The solution is subjected to a color reaction. In interpreting the results of Western blot analysis, IgM requires more than two protein bands corresponding to molecular weights of 41 kDa, 39 kDa, and 24 kDa to exhibit antibody responses, and IgG requires more than five corresponding molecular weights of 93. Protein bands with kDa, 66 kDa, 58 kDa, 45 kDa, 41 kDa, 39 kDa, 30 kDa, 28 kDa, 21 kDa, 18 kDa show an antibody response before they are judged to be positive (see Recommendations for Test Performance) And interpretation from the second National

Conference on Serologic Diagnosis of Lyme Disease. MMWR,

August 11 1995, Vo!.44, No. 31:590-591). m. Western blot analysis is more specific than enzyme immunoassay, but Western blot analysis is still due to other diseases, including syphilis, rheumatoid arthritis, and systemic Lupus Erythematosus (SLE). Etc., and produce a pseudo-5 positive reaction. The Centers for Disease Control and Prevention (CDC) recommends that when a positive reaction is detected by enzyme immunoassay or is undetermined, it should be in the West. The evaluation method for Test Performance and 10 interpretation from the second National Conference on Serologic Diagnosis of Lyme Disease. MMWR, August 11, 1995, VoL 44, No. 31: 590-591. However, 1% of the studies showed that some patients did not meet the test standards prescribed by the US Department of Disease Control for Lyme disease. 'Bacillus burgdorferi can be cultured from samples of these patients. It is because the time of antibody production affects the results of serological diagnosis. For example, after being bitten by an ticks, IgM antibodies are only about 2 to 6 weeks old, and IgG antibodies are separated by 6 to The production started after 8 weeks. The production of antibodies will vary depending on the immunity of the host. Therefore, the sensitivity of the serological test to early diagnosis of Lyme disease is not high (A, R Mares ei al., Journal of Clinical Microbiology, 38 (11): 4239-4241, 2000 Nov.) » There is a "empty window period" in the serum antibody test for Lyme disease, and the culture of skin tissue sections It is quite time consuming (usually 4 to 6 stars, 10 8 stages) and the detection rate is not high (positive rate falls between about 30 and 70%) (HM Feder W α/. (1992), C//« /«/eci. Dii" 15:788-793). As for cerebrospinal fluid The test is more difficult to cultivate and the positive rate is as low as about 5% (E. Asbrink and A. Hovmark, Acta Pathol. Microbiol. Scand., 93:161-163, 1985). In addition, 'B. burgdorferi (5. While the culture of hearing sputum is usually affected by the operator's operational factors, it is necessary to undergo immunofluorescence staining or polymerase chain reaction (PCR) after culturing the bacterium. For further confirmation. PA Rosa and TG Schwan first applied PCR to the detection of Lyme disease (P_A. Rosa and TG Schwan (1989), J. Infect. Dis., 160:1018-1029) 'They revealed The method is to directly detect whether there is a nucleic acid substance of Borrelia burgdorferi (5. ftwrgi/or/en·) in the serum and tissue samples of the patient, and the selected reaction target is in the genome. Gene 2H1 'This gene was later confirmed to encode a surface antigen protein with a molecular weight of 66 kDa (WS Probert ei <3/., /«/eci. 63: 1933-1939, 1995). However, at that time for the gene The designed primer was questioned and could not be It is widely used in the detection of various strains of Borrelia burgdorferi pathogenic strains (5. sensu lato). Studies have shown that flagella (RN Picken (1992), "/. C"«. ^^"〇&_〇/., 30:99-114) and 168 and 238 ribosomes 1^ (1^ 〇8〇11^1 RNA, rRNA) (TM. Richard et al. (1992), Journal of Clinical illusion; 30, 11 (28): 2830-2834) is the preferred detection target. The difference between flagella and 16S rRNA is that there are multiple flagellar genes in the genomic DNA of Borrelia burgdorferi (and 1359196 imrgi/or/m·), so it is suitable for qualitative analysis; and 16SrRNA is in each Borrelia breve (and only one gene copy in genomic DNA, so it may be more suitable for quantitative PCR testing. 5 In addition, unlike flagella or lipoprotein genes with high genetic variability, 16SrRNA genes are It is fairly stable in evolution and it is a single-copy gene in the genomic DNA of the Borrelia burgdorferi pathogenic strain (5. Z>wrgi/or/erz·sensu lato). Therefore, 16S rRNA The gene is suitable for the classification of different subtypes of Borrelia burgdorferi pathogenic strains (5. 10 sensu lato). When the 16S rRNA gene is selected as the target to be amplified by PCR (BL Schmidt β //., Clinical Microbiology Review, p.185-201, Jan. 1997; CL Ling et al. (2000), Journal of Clinical Pathology, 5.3: 94-95; TM Richard et al., Journal of Clinical 15 Microbiology. , 30 ( 11): 2830-2834, Nov. 1992); A Pahl et al.,

Journal of Clinical Microbiology., 37 (6): 1958-1963, Jun. 1999), if the PCR reaction is positive, the Restriction Fragment Length Polymorphism (RFLP) can be further used to identify the Burr Different gene subtypes of the pathogenic strain of Borrelia (5. 20 sensu lato) (D· Liveris fl/., /owrwai 〇/

Clinical Microbiology, 33 (3): 589-595, Mar. 1995); G. Wang et al., Clinical Microbiology Reviews, 12 (4): 633-653, Oct. 1999). In recent years, real-time quantitative polymerase chain reaction (Real time

12 quantitative PCR (hereinafter referred to as real-time quantitative PCR) has been applied to the quantitative analysis of many microorganisms. Compared with traditional PCR, real-time quantitative PCR mainly uses fluorescent substances to detect the entire PCR reaction, and instantly records the amount of amplification of the target DNA, instead of simply detecting the final product after the end of the reaction ( End point detection). There are three types of fluorescent detection modes that are currently used: I. The first type is a method using a Hydrolysis Probe, such as Taqman probe detection, in which the 5' end of the hydrolysis probe is used. It has a fluorescent reporter group and the 3' end has a quencher group. When the primer and the hydrolysis probe are ligated to a target DNA' and a newly synthesized DNA is extended to the position of the probe by a polymerase (p〇lymerase), the hydrolase will probe the probe. The fluorescent reporter group water at the 5' end explains that the fluorescent reporter group will be in a free state and not shielded by the fluorescent quencher group. Therefore, when the fluorescent reporter group is excited (excitati〇n When it emits a fluorescent signal and is recorded by a computer; 第. The first type is a method using a hybridization probe, for example, using fluorescence resonance energy conversion technology (Flu〇rescence Resonance Energy) Transfer, FRET) probe detection method, usually two probes are used, the 3' end of one probe is labeled as a donor fluorescent group (such as FAM), and the other probe The 5' end is labeled to accept an acceptor fluorescent group (eg, LC_Red). When the PCR proceeds to the anneaiing, the two probes simultaneously hybridize to the target DNA, such that the donor fluorescent group and the 1359196 acceptor fluorescent group are adjacent to each other to about 2 to 5 bases apart. The distance between the two bases, at which point the two probes can generate fluorescence resonance energy transfer (FRET) to cause the acceptor fluorescent group to be excited to generate fluorescence; and 5 melon. The third type The type uses a fluorescent dye SYBR Green I specific for double-stranded DNA, which can be incorporated into the amplified PCR product and synchronized by fluorescence intensity detection. Track the progress of the PCR reaction. The fluorescent dye itself is not specific and does not recognize a particular nucleotide sequence, and often results in a false positive result. However, as long as the specificity of the primer is good enough, the fluorescent dye can correctly detect the amplification of the PCR product. It is known that D. Liveris et al. designed a set of primer pairs Pa and P42 for the 16S to 23S rRNA gene spacer of some of the pathogenic strains of Borrelia burgdorferi (5. sensu lato). Liveris ei 15 al. (1995), Journal of Clinical Microbiology, 33: 589-595) 5 and L. Gabriele et al. have targeted the 16S rRNA gene of a part of the Borrelia burgdorferi pathogenic strain (5. sensu lato). A set of primer pairs is designed for PI, P2 (L_Gabriele ei a/., Jowrwa/ 〇/MicroMo/og);, 36 (11): 3355-3358, Nov. 1998). Using the Gene Blast software provided on NCBI website 20 to compare the two sets of primer pairs, respectively, it was found that the pair of primers would have non-specific cross-reaction with other non-pathogenic Borrelia burgdorferi. ). In addition, the difference in melting point of the primers of the two sets of primer pairs was 4 ° C and 7 ° C, respectively, and thus was not suitable for use in real-time quantitative PCR. '

14 1359196 M. Schwaiger et al. designed a set of primers for a fragment of a chromosomal gene encoding a flagellin protein, one of the pathogenic strains of Borrelia burgdorferi (and Zmrgi/or/en·sensu lato). For FlaFlA, FlaRl and a fluorescent TaqMan probe (FlaProbel) (M. 5 Schwaiger et al. (2001), Clinical Microbiology and /w/ec as ms Diseiwe, (7): 461-469). Although the use of this primer with the probe can increase the specificity of detection, the method disclosed by M. Schwaiger et al. cannot detect the genus Borrelia (5. Japcmica) and thus cannot be applied to all Borrelia burgdorferi. Inspection of pathogenic strains (and sensu 10 lato). C. Rauter et al. designed an internal marker primer for the outer surface protein A gene (os/M) of the pathogenic strain of Borrelia burgdorferi (β. sensu lato). ) with a fluorescently labeled probe (HybProbe) (C. Rauter)

15 et al., Journal of Clinical Microbiology, 40 (1): 36-43, Jan. 2002). A method disclosed by C. Rauter et al. has the property that a melting point analysis can directly distinguish one of the Borrelia burgdorferi gene strains (5. sensu stricto), B. burgdorferi (5. garim·/) and The three gene strains of Borrelia oxysporum (5. fl/ze/ζϊ) have a melting point of about 5 °C. However, the combination of the primer and the fluorescent hybrid probe could not detect the Borrelia spirulina (five force), so the method disclosed by C. Rauter et al. cannot be applied to all Borrelia burgdorferi. Test for pathogenic strains of bacteria (Naga·8^r/^^sensu lato) Based on the above, provide a test for quantitative and qualitative identification of nucleic acids

15 1359196 The system has an urgent need to supply rapid, sensitive and specific tests for Lyme disease and to address the problem of serological examination of Lyme disease. SUMMARY OF THE INVENTION 3 5 SUMMARY OF THE INVENTION Thus, in a first aspect, the present invention provides a primer pair for detecting a pathogenic strain of Borrelia burgdorferi (5orre/ia sensu lato) a 16S rRNA gene of Borrelia species, ???) is designed as a forward primer and a reverse primer, which is basically composed of the following sequence Identification number: The nucleotide sequence shown in Figure 1, and the reverse primer is basically composed of a nucleotide sequence as shown in the following sequence identification number: 2: Forward introduction 15 5 '-cgtaaggggcgagcgttgttc-3, (sequence identification number: 1)

Reverse primer 5'-cagaagttcgccttcgcctcc-3' (sequence identification number: 2) In the second aspect, the present invention provides a test kit 20 for detecting a pathogenic strain of Borrelia burgdorferi (sensu lato) (diagnostic kit) ), which comprises a set of primer pairs designed according to the 16S rRNA gene of the Borrelia burgdorferi pathogenic strain sensu lato). The primer pair comprises a forward primer and a reverse primer, and the forward primer is basically It consists of a nucleotide sequence as shown in sequence identification number: 1, and the reverse primer consists essentially of a nucleotide sequence as shown in Sequence Identification 16 1359196 No.: 2. In a third aspect, the present invention provides a method for detecting whether a body has Lyme disease in vitro, comprising: using a biological sample taken from an individual suspected of having Lyme disease, A set of 5 pairs as shown above is subjected to a polymerase chain reaction to detect the presence of a 16S rRNA gene of a Borrelia burgdorferi pathogenic strain (and sensu lato) in the sample. The presence of the 16S rRNA gene of the spirochete pathogenic strain (5orre/k 6ttrgi/〇r/en·sensu lato) is a characteristic of Lyme disease. The above and other objects, features, and advantages of the present invention will become apparent from Display using primer pair P1, P2 and Borrelia burgdorferi 15

20 hrgi/or/) B31 genomic DNA (ATCC 35210D) is formulated at different concentrations (20 ng/pL, 2 ng/μί, 200 pg/pL, 20 pg/yL, 2 pg//iL, 0.2 pg/ The standard solution of pL) is used to perform a quantitative standard curve after real-time quantitative PCR, where the abscissa indicates the log concentration of each standard solution, and the ordinate indicates the heat of each standard solution into the log phase. Figure 2 shows the TWLYM-1 (SEQ ID NO: 1), TWLYM-2 (SEQ ID NO: 2), and genomic DNA of Borrelia burgdorferi B31 using the primers of the present invention ( ATCC 35210D) Prepare standard solutions of different concentrations (20 ng//iL, 2 ng/pL, 200 pg/μί, 20 pg/μL, 2 pg/μί, 0.2 pg/pL) for real-time quantitative PCR A quantitative standard curve is obtained in which the abscissa indicates the logarithmic concentration of each standard solution' and the ordinate indicates the number of thermal cycles in which each standard liquid enters the log phase; FIG. 3 is an electrophoresis film diagram showing the use of primer pair pi, P2 And different DNA samples for real-time quantitative PCR production of pCR Analysis results of agarose gel electrophoresis, wherein: diameter 1 is a DNA ladder marker; diameter 2 is a negative control (no DNA template); diameter 3 and diameter 4 are respectively Bacillus licheniformis iwg r/en) standard solution prepared by B31 genomic DNA (ATCC 35210D) (20 ng/pL, 2 ng//tL); path 5 is one from the fourth group of suspected Lyme disease cases One of the patient's vesicle fluid samples; and the diameters 6, 7, and 8 are three subcultured standard strains (ATCC 35210, ATCC 51567, and ATCC 51383); Figure 4 is an electrophoretic film diagram showing the use of this The result of the agarose gel electrophoresis analysis of the PCR product obtained by real-time quantitative PCR on TWLYM-1, TWLYM-2 and different DNA samples, wherein the diameter 1 is the DNA ladder mark and the diameter 2 is the negative control group ( Does not contain any DNA template); Paths 3, 4, 5 and 6 are prepared at different concentrations (200 pg/pL, 20 pg/pL, 2 pg/) using genomic DNA of Borrelia burgdorferi B31 (ATCC 35210D). /iL, 0.2 pg//iL) standard solution; and path 7 and diameter 8 are from the first group of Lyme disease cases a serum sample of the patient; and FIG. 5 is an electrophoretic film diagram showing the PCR product obtained by performing real-time quantitative PCR using the primer of the present invention on TWLYM-1, TWLYM-2, and different DNA template samples, First, the restriction enzymes (restricti〇n endomidease) were used for cleavage, and then electrophoresis analysis was carried out by polyacrylamide gel electrophoresis (PAGE), in which the sputum was the genomic DNA of Borrelia burgdorferi B31 (ATCC). 35210D) Standard solution (200 pg//iL) (positive control group); diameters 2, 3, and 4 are serum samples from patients in the fourth group of suspected Lyme disease cases; and path 5 DNA ladder mark (100-1000 bp). C Embodiment 3 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The serological examination of Lyme disease has a problem of empty window period. Only 67 4 〇/〇 of Lyme disease patients will detect lgM antibody within 60 days, and it is not easy to be combined with other Disease differentiation, so 'how to detect Lyme disease quickly and accurately is particularly important. The immediate quantitative polymerase chain reaction is time-saving and saves a lot of time compared to antibody detection or skin culture. Therefore, the applicant is committed to developing rapid detection methods in this area. Unlike flagella or lipoprotein-encoding genes with high variability, the 16S rRNA gene is fairly stable in evolution and it is in the pathogenic strain of Borrelia burgdorferi (Borre/ia Zmrgdor/en·sensu lato) There is only one copy of genomic DNA, so it can be used as a target for quantitative analysis. In the present invention, the applicant uses a 16S rRNA differential dendrogram of each prokaryote to design a primer which can be used for PCR reaction of the 16S rRNA gene of Borrelia burgdorferi, based on the designed difference in the 3' end of the primer. The principle of sex (3, if it is a non-corresponding nucleotide 'there is no starting point for replication), to 1359196 causes non-pathogenic strains to fail to carry out polymerase chain reaction. Thus, pathogenic to Borrelia burgdorferi The 16S rRNA gene of the sexual strain plus rgdor/eW sensu lato), the applicant designed a set of specific primer pairs in the present invention, namely TWLYM-1 and TWLYM-2, and first applied to test serum samples and A vesicle fluid sample for the presence of the pathogenic strain sensu lato) of Borrelia burgdorferi. It has been experimentally confirmed that the primer pairs according to the present invention can detect all pathogenic strains and exclude the possibility of cross-reaction of non-pathogenic strains. 10 Moreover, the pair has the following characteristics: the design of the primer is highly matched; the interference between the inner primer dimer and the inter primer dimer is low; and the melting point of the primer is less than 1°. C. Thus, the pair of primers according to the present invention can be applied to a method for detecting whether a body has Lyme disease in vitro, the method comprising: taking 15 from an individual suspected of having Lyme disease Biological sample, using a set of primer pairs as described above for polymerase chain reaction, to detect the presence of a pathogenic strain of Borrelia burgdorferi in the sample (5orre(6) △Mrgi/or/en·sensu lato The 16S rRNA gene of the Borrelia burgdorferi pathogenic strain rym·sensu lato) is characterized by the presence of the Lyme disease. In one embodiment, the biological sample is selected from the group consisting of blood, serum, plasma, vesicle fluid, cerebrospinal fluid, pericardial fluid, joint fluid, ascites, lymph nodes, saliva, and urine. In a more preferred embodiment of the invention, the biological sample is serum. In another 8 1359196 immunosorbent assay of the invention, ELISA): anti-B. burgdorferi (ELISA analysis of Borre/ia IgM/IgG) in biological samples (eg, serum samples) taken from suspected Lyme disease cases Refer to the general clinical testing procedure (see, for example, κ. 5 Hansen et al. (1988), Journal of Clinical Microbiology, 26: 338-346! M. Karlsson (1990), Journal of Clinical 28: 2148-2150 ) and using the Lyme Borreliosis ELISA Kit Code No. K0416 (Dako Cytomation, Danmark). 10 B. Western blotting: Biological samples taken from suspected Lyme disease cases (eg, serum samples) Western blot analysis of anti-B. burgdorferi IgM/IgG is based on general clinical testing procedures (see, for example, SM Elizabath (1995), Journal of Clinical Microbiology., 33 15 (2): 419-427 I F. Dressier et al. (1993), Journal of Infectious

Dheases., 167:392-400 and Recommendations for Test Performance and interpretation from the second National Conference on Serologic Diagnosis of Lyme Disease. MMWR, dwgwsi 7/, 79P5, and using MarDx 20 Borrelia Burgdorferi IgM/IgG Marblot Strip Test System ( Trinity: FDA Cleared, Product Code: 40-2065M/G, USA). C. Agarose gel electrophoresis: 1. Prepare 2% agarose gel: 23 8 1359196 4 g of phospholipid powder (LE analytical grade agarose, Cat.

No. V3125, Promega) was dissolved in 200 mL of IX TBE (Cat. No. MRB-0003, Maxim Biotec, Inc.) and then the mixture was heated in a microwave oven for 3 minutes to completely dissolve the powder and applied to the electromagnetic stirrer. The mixture was stirred and mixed for about 10 minutes to cool the mixture to about 40 to 42 ° C, followed by 10 / aL ethidium bromide (Etium bromide, EtBr, 0.5 mg / L, Sigma). After the mixture is uniformly mixed, the mixture is poured into a pre-prepared mold to solidify the mixture to form an electrophoretic film. 2. Electrophoresis operation:

10 The formed 2% agarose film was placed in an electrophoresis tank EC 360M (E-C electrophoretic gel system), followed by the addition of IX TBE. Take 2 / xL of loading dye buffer (containing 50 ° / 〇 glycerol, 12.5

mM Tris-HCl pH 8.0, 60 mM EDTA pH 8.0, 0.01% bromophenol blue, 0.01% Xylene cyanol FF, add 15 μί of PCR product to be tested, to be mixed evenly It was then added to the groove of 2% agarose film. Another 2 pL of nucleic acid molecular weight marker (DNA molecular weight marker, 100 bp ladder, 100-1000 bp, GeneSura Laboratory, USA) was mixed with 2 gL of dye buffer and 8 / x L of TBE buffer 20 solution. After being uniformly mixed, it was added to a groove of 2% agarose film to perform simultaneous control electrophoresis with the PCR product to be tested, and the current was set to 1 〇〇v (EC250-90 power supply), and the electrophoresis was carried out for 30 to 40 minutes. . The electrophoresed 2% agarose film was placed on a UV transilluminator (Vilber Lourmat) and stored and imaged as a camera.

24 Analytical System (Photo-print Photodocumentation system, Vilber Lourmat) to observe and photograph the results of electrophoresis. Thereafter, the molecular weight of the PCR product was analyzed using PhotoCaptMW/Biogene software. D. Purification and sequencing of the PCR product: The PCR product after 2% agarose gel electrophoresis was excavated and recovered from the bright band by a knife under a UV lamp (UV transilluminator M-26; 260 nm). Then, it was purified by Gene-SpinTM 1-4-3 DNA Extraction Kit manufactured by Protech Technology Enterprise Co., Ltd. The recovered agarose was mixed with an equal amount of binding solution, placed at 60 ° C for 10 minutes, the mixed liquid was aspirated and transferred to a spin-spin column, and centrifuged at 13,000 rpm. It lasted 1 minute. I abandon the solution' and add 500 pL of the binding solution, centrifuge at 13,000 rpm for 1 minute, discard the filtrate, and add 700 pL of washing solution to centrifuge at 13,000 rpm for 1 minute. Discard the filtrate, transfer the residue to a clean centrifuge tube, add 50 g of double distilled water, centrifuge at 13,000 rpm for 5 minutes, collect the filtrate (DNA) and use ABI BigDye Terminator reagent and PERKIN ELMER The ABI PRISMTM 377 DNA Sequencer was used for sequencing, and the resulting sequencing results were analyzed using DNA Star kit software (DNASTAR, Inc.). E. Restriction fragment length polymorphism (RFLP): The enzyme digestion of the PCR product was carried out by selecting 1359196 Restriction enzyme Z/zTzyi using DNAstar MapDraw software (DNASTAR, Inc.). Enzyme digestion). The PCR product to be tested was treated with the nucleic acid restriction enzyme i (Promega Cooperation) at 37 C for 3 hours, and the reaction conditions were as follows: 5 1 〇 ML PCR product was mixed with 2 / iL of 10X Multi-Core Buffer (Promega) and restriction enzyme / Λ · η / (5 units), then make up the volume to 20 with water. After 3 hours of reaction, electrophoresis was carried out with 8% Polyacrylamide gel, and a nucleic acid molecular weight marker (25-bp marker, Promega Cooperation) was used as a control electrophoresis. After the end of electrophoresis 10, the gel was stained with a brominated ethyl bond and photographed under a UV lamp (uv transilluminator M-26; 260 nm).生物. Biomaterials: A, standard strain and genomic DNA: In the following examples, use the American Type Culture Collection 15 Center (ATCC, RO. Box 1549, Manassas, VA 20108, USA) Bo

Borrelia burgdorferi B31 (ATCC 35210), Borrelia stipitis CIP 103469 [P12] (ATCC 51567) and Borrelia bulgaricus CSo/re/ία gflnm·/) CIP 103362 (ATCC 51383) were used as standard strains. 20 In addition, genomic DNA (ATCC 35210D) derived from Borrelia burgdorferi ΟδοηΆ'α B31, purchased from the American Type Culture Collection Center, was used in the following examples at an original concentration of 200 ng/j^L, The total volume is 50 μί. Prior to the experiment, sterilized water was used for 10-fold serial dilution to prepare different concentrations (20 26 8 1359196 ng//xL, 2 ng//iL, 200 pg/μί, 20 叩Machine, 2 pg/吣, 0.2 pg/ML) standard solutions, and stored at _2〇. Keep your spare. B. Culture of standard strains: According to the growth conditions provided by the American Type Culture Collection Center, 5 strains of each of the 仏 仏 ' ' 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中The culture temperature was 37C' and the culture temperature of Borrelia burgdorferi (square i^e/z·/) and Borrelia burgdorferi (5orre/i_a garim.z) was 35 °C. The standard strain was cultured in a 25T flask containing BSK-H medium (Sigma) and 6% rabbit serum (Sigma). In order to avoid the growth of bacteria during culture, rifampin (50 mg / L), phosphomycin (100 mg / L) and amphotericin B (Amphotericin Β) can be added to the culture medium. ) (2.5 mg/L). When the medium appeared turbid, the cells were collected by centrifugation and seeded into a 25T flask containing the medium to make the bacteria more adaptable to the growth environment of the medium. Then, 15% of the glycerin was added to the medium containing the bacteria 15, and after being dispensed into a 1.5 mL vial, it was stored in a -7 CTC refrigerator for use. Dish·Collection of serum samples and vesicle fluid samples for Lyme disease: The routine experimental diagnosis procedure for Lyme disease is: first perform an individual serum test or perform skin section culture. The serum antibody test is performed by an ELISA assay as described in 20, and the sample is positive or indeterminate and confirmed by Western blot analysis as described above. In the following examples, serum samples from 62 confirmed cases of Lyme disease were selected from the serological examination samples of 935 suspected Lyme disease individuals collected by Dr. Cai Jijun of Kaohsiung Medical University for infection, 27 1359196 These include: the first group is the Western blot method IgM positive, IgG negative serum samples, a total of 43; the second group is igM and IgG are positive serum samples, a total of 5; and the third group is IgM negative, There were 14 IgG-positive serum samples. 5 A fourth group, which is a serum sample of IgM and IgG negative reactions but highly suspected to be suspected to be Lyme disease, has a total of 25 serum samples.

Further, a vesicle fluid sample used in the following examples was obtained by withdrawing the needle from the superficial skin of the patient. Example 1 Polymerase chain reaction (PCR) of 16S DNA gene of Borrelia burgdorferi pathogenic strain 6wrp/or/en' 10 sensu lato) and real-time quantitative PCR A. Design of the primer: download all pathogenic strains of Borrelia burgdorferi pathogenic strain 15 sensu lato) from the NCBI website [including Borrelia burgdorferi

One gene strain (5· sensu stricto), borreliosis of B. bulgaricus (5· and Borrelia genus (5. and B. ya/?om_ca) nucleosides of 16S rRNA gene Acid sequence, as well as the nucleotide sequence of the non-pathogenic regression of Borrelia burgdorferi (5· recM/rewih), Borrelia burgdorferi (Where 20 and so on) 16S rRNA gene. Selection of Borrelia burgdorferi B31 The complete genome [NCBI's accession number is NC_001318] is used as a reference sequence for sequence alignment and is performed by DNA star MegAlign software (DNASTAR, Inc.).

28 1359196 The ordering of the 16S rRNA genes of the CSorre/(4) strain of Borrelia is described to confirm the conservative regions and variant regions within the 16S rRNA gene of these strains. Select about 40 to 50 pathogenic Borrelia burgdorferi-specific gene fragments from the gene tree of 16S rRNA 5 containing all bacteria, spirochetes, chlamydia and mold, and use DNAstarMegAlign software. To rank the conserved regions within the 16S rRNA gene of these gene fragments and the Borrelia strains identified above, and to find three nuclear seedling residue positions corresponding to the 16S rRNA gene, respectively, 10 400-430 , 500~560 and 710-770 areas. 15

Primers for these three regions were designed using three sets of software: DNA star PrimerSlect (DNASTAR, Inc.), Light Cycler Primer and Probe Design (Roche), and TM calculator (TM utility vl. 3; Roche). The designed primer must be tested with DNA star PrimerSlect software for its structural stability, inner primer dimer and inter primer dimer, and then to the NCBI website for gene BLAST analysis. '俾 to confirm the possibility of no other cross-reactions, and to the human chromosome gene database for comparison, to eliminate the possibility of corresponding to human genes. 20 Thus, a set of primer pairs having the nucleotide sequences shown below were obtained TWLYM-1 and TWLYM-2: forward primer TWLYM-1 5'-cgtaaggggcgagcgttgttc-3' (sequence identification number: 1)

29 1359196 Reverse primer TWLYM-2 5'-cagaagttcgccttcgcctcc-3' (SEQ ID NO: 2) wherein TWLYM-1 corresponds to the nucleotide residue of the 16S rRNA gene of Borrelia burgdorferi hrgcbr/m·) B31 Position 532 to 5 552 ' and TWLYM-2 corresponds to the NK 11 of the Borrelia burgdorferi~cut ^/:/^/) 331 11^ into the nucleotide residue position of the gene 738 to 718 » In addition, for this A comparison of the primer pairs designed by the invention was made in L. Gabriele ei β/. (1998), /owrwa/ 〇/ C/Wca/ 10 illus;, 36 (11): 3355-3358 Genus-specific primer pair P1 and P2 designed to have the nucleotide sequence shown below for a gene strain of Borrelia burgdorferi (where ^mrgi/or/en·sensu stricto) : Forward introduction P1 15 5'-acgctggcagtgcgtcttaa-3' (sequence identification number·· 3)

Reverse primer P2 5'-ctgatatcaacagattccaccc-3' (SEQ ID NO: 4) wherein P1 corresponds to the nucleotide residue position of the 16S rRNA gene of a gene strain of Borrelia burgdorferi (5. sensu stricto) 2 sets 39 to 58, and the P2 line corresponds to the nuclear acid residue position 708 to 687 of the 16S rRNA gene of one of the Borrelia burgdorferi gene strains (and 6 wlgdor/en sensu stricto). B. Extraction of genomic DNA: Extraction and purification of genomic DN A from each standard strain or clinical sample 30 can be performed using the following three commercial purification kits: (i) DNA isolation kits for cells and tissues (DNA) Isolation Kit for Cells and Tissues, Roche, Cat. No. 1814770), GFX Genomic Blood DNA Purification Kit (Pharmacia, Cat. No. 27-9603-03) and MagNA Pure LC DNA Separation Kit Group III (MagNA Pure LC DNA Isolation Kit III, R0Che, Cat. No. 3264785). In this experiment, the extraction and purification of genomic DNA from each standard strain or clinical sample was performed using the GFX Genomic Blood DNA Purification kit (Pharmacia, Cat. No. 27-9603-03). . C. Polymerase chain reaction (PCR): 1. Use primer pair PI and P2: Use the commercial kit Fast-RunTM Taq Master Kit (Protech) for PCR' operating conditions: at 95 ° C Denature took 45 cycles for 45 cycles; at 94 °C, the denaturation lasted 30 seconds; at 55. (: Next, primers for 30 seconds; at 72 °C, elongation is extended for 90 seconds; finally at 72 ° for 10 minutes. When Bordetella burgdorferi Zmrgi/or/ En·) B31 genomic DNA (ATCC 35210D) was used as a template, and the PCR product thus obtained was a dsDNA of approximately 670 bp. 2. Using the primer pair TWLYM-1 and TWLYM-2: PCR was performed using the commercial kit Fast-RunTM Taq Master Kit (Protech) under the conditions of: denaturation at 95 ° C for 5 minutes, 1359196 45 cycles; at 94 ° C, the denaturation lasted 30 seconds; at 56 ° C, the primers were subjected to primers for 30 seconds; at 72 ° C, the elongation was extended for 60 seconds; Finally, at 72 ° C, it lasted 10 minutes. When Borrelia burgdorferi (5orre/i'ii B31 gene 5 group DNA (ATCC 35210D) and two subcultured standard strains of Borrelia lyrxium α/ze) (ATCC 51567) and Gary When the genomic DNA of spirochete garim·!) (ATCC 51383) is used as a template, the PCR product thus obtained is a dsDNA of approximately 207 bp» In addition, due to part of Borrelia burgdorferi (5orre/iiZ yapon/cfl) 10 One nucleotide residue "a" will be added at the nucleotide residue position 559 of the 16S rRNA gene. If the genomic DNA of Borrelia spirulina is used as a template, one will get about 207~208. Bp dsDNA. D. Real time quantitative PCR: 15 This experiment uses LightCycler FastStart DNA Master

SYBR Green I commercial kit (R〇che, Cat. No. 3 003 230) and LightCycler 2.0 instrument (Roche) for immediate genomic DNA of standard strains or clinical samples prepared as described in item B above Quantitative PCR. The 20 setting methods for material preparation methods and reaction operating conditions for real-time quantitative PCR are based on the instruction manual provided in the LightCycler FastStart DNA Master SYBR Green I commercial kit and modified slightly. The hot start reaction mix (Roche, Cat. No. 3 003 230) used in this experiment contains FastStart Taq DNA polymerase (polymerase) and SYBR Green I fluorescent dye 〇 32 1359196

FastStart Taq DNA polymerase is inactive at room temperature because heat-labile blocking groups are bonded to certain amino acids of the enzyme at 95 ° C. The blocking groups are removed for at least 10 minutes to allow the enzyme to exhibit activity. The SYBR 5 Green I dye is specific for the DNA of the double helix, and as the polymerase chain reaction progresses, as the PCR product increases, the fluorescence intensity increases. When the real-time quantitative PCR uses SYBR Green I format for UghtCycler, this technology can distinguish the properties of the product according to the molecular weight and G+C content. Compared with the traditional electrophoresis analysis, the principle of molecular weight size 10 can only be distinguished. This technique can more accurately Analysis of PCR Products First, 'FastStart Taq DNA polymerase (polymerase) was added to a vial containing a FastStart Reaction Mix SYBR Green I, and gently mixed by a pipette to obtain a heat. The hot start reaction mixture was started and stored in the dark at 15 °C for use.

Add 2.4 μM MgCl2 (4 mM) to a 1.5 mL PCR tube, 1 μΐ each of the forward and reverse primers (0.5 μΜ each), 7 μΐ water (PCR grade), and 2 μΐ hot start The hot-start reaction mixture is gently mixed with a pipette to form a master mix. The resulting master mix is added to a pre-cooled mixture. In a LightCycler capillary (Roche), a 5 μΐ DNA template was added. The capillary was centrifuged at 3,000 rpm for 5 seconds, then the capillary was placed into the rotor of the LightCycler instrument.

33 1359196, and according to the following operating procedures for real-time quantitative pCR, where the positive control is the use of Borrelia burgdorferi

Zwg and r/en) B31 genomic DNA (ATCC 35210D) was used as a DNA template and formulated to different concentrations for the production of a quantitative standard curve, 5 and the negative control contained all PCR components but No DNA template. 1. Procedure for using P1 and P2 primers: Pre-incubation of the FastStart Taq DNA polymerase (pre-incubati.on) and denaturation of the DNA template at 95 °C for 10 1 min, followed by 10 1 min. The amplification reaction was carried out (95 ° C for 2 seconds, 58 ° C for 1 second, and 72 ° C for 29 seconds for 45 cycles). 2. Procedure for using TWLYM-1 and TWLYM-2 when using primers: 15

The pre-incubation of FastStart Taq DNA polymerase and the denaturation reaction of the DNA template were carried out at 95 ° C for 1 minute, followed by amplification reaction (95 ° C for 2 seconds, 62 ° C for 1 second, 72 ° C for 10 seconds). 45 cycles)) In addition, the number of bacteria in a test sample can be calculated by the following formula: = quantity [replica / Vl] 6 乂 1023 [replica / 111 〇 1] < concentration [^ / ^ 1 ^ & quot MW [g/mol] Concentration: DNA concentration of a Borrelia burgdorferi strain sample calculated by aligning the quantitative standard curve 20: molecular weight of genomic DNA of Borrelia burgdorferi strain; calculation formula: MW= 920000 bps X 660 Dalton/bp 34 Ε· Melting point analysis method After the completion of the real-time quantitative PCR amplification reaction, the obtained PCR product was subjected to melting curve analysis by LightCycler instrument. 95 ° C for 5 seconds, 60 ° C for 30 seconds, then heated to 95 ° C at a rate of 0.1 ° C per second, and continuously detect the fluorescence reaction, and finally at 40 ° C to make the LightCycler instrument rotor Cooling with the reaction tank lasted for 30 seconds. Results: A. PCR efficiency: When using different concentrations (2〇ng/μΐ, 2 ng/μΐ, 200 pg/μΐ, 20 pg/μΐ, 2 pg/μΐ, .0.2 pg/μΐ) The genomic DNA of Borrelia burgdorferi B31 (ATCC 35210D) was used as a DNA template standard solution, and the primers pair P1 and P2 were used for real-time quantitative PCR using SYBR green I mode. A curve of 5 was obtained (data not shown). From the 5-graph, it can be observed that when the number of thermal cycles of the PCR increases, the detected fluorescence absorbance value also increases, and the standard solutions of different concentrations will enter the index successively. The log phase is reached and finally reaches the plateau phase. Taking the log concentration as the horizontal axis' and the number of thermal cycles in which the standard liquid enters the exponential increase period (according to the (5 graph)) as the vertical axis, a graph can be obtained. Quantitative standard curve shown in 1. This quantitative standard curve is via

The LightCycler software analysis yielded a slope of 4.008 ± 0.4. The slope was then converted by the formula "PCR efficiency 10 = 10·1/4^Λ", and the PCR efficiency was 1.776 ± 0.1. 1359196 When using the primer pair TWLYM-l, TWLYM-2, and using the same DNA template standard solution for real-time quantitative PCR using the ffiSYBR 8 surface mode, a -5 graph is also obtained (data not shown) The logarithm of the concentration of the standard solution (Log(10)eentfatiGn) is mixed, and the number of thermal cycles in which the standard solution enters the exponential increasing period (calculated according to the 5-graph) is obtained as a vertical axis, as shown in FIG. Quantitative standard curve. The quantitative standard curve was analyzed by LightCycler software to obtain a slope of 3.738±0.18. The slope was then converted by the formula “pcr efficiency e=1〇-i/ oblique φ value” to obtain a PCR efficiency of 1.852. ±〇.〇2. From the above results, it can be seen that a good linear relationship can be obtained by using the primer pair P1 and P2 and the primer pair TWLYM-1 and TWLYM-2, respectively, to obtain a good linear relationship. (: Ruler efficiency and from the analysis of coefficient of variation (data not shown), the results of TWLYM-1 and TWLYM-2 using the primer according to the present invention are better. 15 B. Melting point analysis

When using genomic DNA (ATCC 35210D) of Borrelia burgdorferi ftwrgi/o/y'(7)·) B31 as a DNA template' and using a primer pair? When 1 and P2 were used for real-time quantitative PCR using the SYBR green I mode, a 670 bp PCR product was obtained. The PCR product was analyzed by melting point, and it was observed that 20 had a peak at 87.44 ° C. The inter-assay coefficient of the peak was 0.25%, and the intra-assay coefficient of variation (intra-assay) was analyzed. The coefficient) is 0.05%. As for the primer dimer, the refining point is about 74 to 82 °C. Therefore, in order to avoid the influence of the primer dimer on the quantitative accuracy, in the quantitative curve, the temperature of the 36 debt can be increased to 83 ° C, and the measured absorbance of the fluorescence is derived from the PCR product. The interference factor of the primer dimer can be solved. In addition, when the genomic DNA of three subcultured standard strains (ATCC 3521〇, ATCC 51567 and ATCC 51383) was used as a DNA template for real-time quantitative PCR, the melting points of the PCR products of the three standard strains were measured as 87.48 ° C, 87.46 ° C and 87.42. (:, therefore, it is not possible to distinguish the three standard strains by melting point analysis. When using the primer pair TWLYM-1, TWLYM-2, and using the same DNA template for real-time quantitative PCR using the sybr green I mode, A PCR product of 207 bp was obtained. The melting point of the PCR product was observed to have a peak at 82.42 ° C. The inter-assay coefficient of variation of the peak was 0.19%, and the intra-assay coefficient of variation was 〇〇 5%. As for the melting point of the primer dimer, it is located at about 81.23 ° C, which is quite close to the melting point of the PCR product, and it is highly likely to affect the accuracy of the quantification. Therefore, in order to avoid the occurrence of the dimer of the primer, the applicant It has been found that the primer's annealing time is set to 1 second, the primer concentration is reduced to 〇3 μΜ, and the magnesium ion (Mg++) concentration is reduced to 3 mM. These methods can avoid primer dimers. In addition, when the genomic DNA of three subcultured standard strains (ATCC 3521〇, ATCC 51567 and ATCC 51383) was used as a DNA template for real-time quantitative PCR, the P of the three standard strains The melting point of the CR product was 82.31, 〇, 82.67. (3, 82.45. 〇, therefore, the melting point analysis could not be used to distinguish the three standard strains. 1359196 C. Agarose gel electrophoresis analysis and restriction of PCR products Enzyme fragment length polymorphism analysis (RFLP): Figure 3 is an electrophoresis film showing the use of primer pairs pl, p2 and different DNA samples for PCR products obtained by real-time quantitative 5 PCR using SYBR green I mode. Agarose gaze electrophoresis (agar〇se gei electrophoresis) analysis results, including: ladder mark; diameter 2 is negative control (negative control) (excluding any DNA template); diameter 3 and diameter 4 are respectively Standard solution prepared from genomic DNA of B. sp. B31 (ATCC 35210D) (20 ng//iL, 2 10 ng/pL); path 5 is a pouch from the fourth group of patients suspected of Lyme disease The bubble sample; and the diameters 6, 7 and 8 are three subcultured standard strains respectively (into Ding: 35210, eight 7' <: € 51567 and into 1 ' (: € 51383).

Figure 4 is an electrophoresis film showing agarose gel of PCR product obtained by real-time quantitative PCR using SYBR green I mode using TWLYM-1, TWLYM-2 and different DNA samples of the present invention. The results of electrophoresis analysis showed that the diameter 1 was the DNA ladder mark; the diameter 2 was the negative control group (excluding any DNA template); the diameters 3, 4, 5 and 6 were respectively using Borrelia burgdorferi ftwrgi/or/eri) B31 Genomic DNA (ATCC 35210D) was formulated into standard solutions of different concentrations (200 pg//xL, 20 pg/pL, 2 20 pg/μί, 0.2 pg/juL); and diameter 7 and diameter 8 were respectively first Serum samples from patients in the Lyme disease case. Figure 5 is an electrophoresis film showing the use of the primers of the present invention for TWLYM-1, TWLYM-2 and different DNA template samples for PCR production of 38 V using real-time quantitative PCR using the SYBR green I mode. The results of electrophoretic analysis were performed by restriction endonuclease followed by polyacrylamide gel electrophoresis (PAGE), in which the diameter 1 was the genomic DNA of Borrelia burgdorferi B31 ( ATCC 35210D) standard solution (200 pg/pL) (% control); diameters 2, 3 and 4 are serum samples from patients in the fourth group of suspected Lyme disease cases; and path 5 Enter the ladder mark (100-100(^?) for the £1). The electropherogram is calculated by 1>11 with 〇(:邮)^曹;8丨(^1^Software to calculate the molecular weight of the product. Type analysis (RFLP) usually performs gene mutation analysis on different subtypes, serotypes or isolates of the same or the same strain. The results of this graph show that the three are from Serum samples from patients in the fourth group of suspected Lyme disease cases One of the gene strains sensu stricto) has the same cut fragment. Therefore, it is inferred that the three serum samples from patients in the fourth group of suspected Lyme disease cases contain Borrelia burgdorferi. _ _ _ sensu stricto. D. Sequence analysis of PCR products: using the primer pair TWLYM-1 and TWLYM-2 to the pathogenic strain of Borrelia burgdorferi 6Mrgdor/m·sensu lato) The genomic DNA of the three gene subtypes was subjected to real-time quantitative PCR using the SYBR green I mode, and the thus obtained PCR product was subjected to sequence analysis to obtain the following scoring: 1 · One gene strain of Borrelia burgdorferi (5. sensu stricto) 1359196 5 cgtaaggggc cggatatata gt tggaaact gtaagggtgg act tctg gagcgt tgt t agtctatgca atatgtctag aatctgt tga cgggattatt taaaatacca agtctgatag tatcagaaag gggcgtaaag cagctcaact aggaagt tag aataccggag ggtgagtagg gtggacctat aat t tctggt gcgaaggcga (sequence identification number: 5)

Gggcgtaaag ggtgagtagg cagctcaact gtggaactat aggaagt tag aat t tctggt aataccggag gcgaaggcga (SEQ ID NO: 6) 10 cgtaaggggc gagcgttgtt cgggattatt cggatatata agtctatgca taaaatacca gttggaaact atatatctag agtctgatag gtaagggtgg aatctgttga tatcagaaag act tctg 3. Borrelia genus

cgtaaggggc cggatatata gttggaaact gtaagggtgg gagcgttgtt agtctatgca atatgtctag aatctgttga cgggattatt taaaatacca agtctgatag tatcaggaag gggcgtaaag cagctcaact aggaagttag aataccggag ggtgagtagg gtggagctat aat tcctggt gcgaaggcga acttctg (SEQ ID. No: 7) primers of Example 2. TWLYM-1 and TWLYM-2 compared to PI and P2 primers The utility of clinical detection in Lyme disease is to evaluate the primer pair TWLYM-1 and TWLYM-2 of the present invention, compared to L. Gabriele ei alpha (1998), /oMrwa/o/C/im_ca / Micro6io/og_y, 36 (11): The utility of the primer pair PI and P2 disclosed in 3355-3358 for clinical detection of Lyme disease, using the following four groups of serum samples for S YBR green I mode Real-time quantitative PCR: 40 1. The first group was Western ImgM positive, IgG negative serum samples, a total of 43; 2. The second group was serum samples positive for both IgM and IgG, a total of 5; The third group was IgM-negative, IgG-positive serum samples, a total of 14; and 4. The fourth group was serum samples of IgM and IgG-negative responses but highly suspected to be suspected to be Lyme disease individuals. There are 25 in total. A. Effect of primers on clinical detection of P1 and P2 in Lyme disease Table 1 shows that serum samples of confirmed cases of Lyme disease were detected using real-time quantitative p CR using SYBR green I mode using different primer pairs. Results When the PI and P2 were tested by primers, 4 of the 11 confirmed cases of Lyme disease were positive, and the positive rate was 36.3%. The PCR products of the four positive samples were further confirmed by melting point analysis and agarose gel electrophoresis analysis, wherein the average melting point temperature of the PCR products of the four positive samples was 86.71±0.5311 C' as determined by melting point analysis. At 87.44 ° C measured by the PCR product of genomic DNA of jgorre/k B31 (ATCC 35210D), there is a difference of 0.67 ° C between the two, and when analyzed by agarose gel electrophoresis To analyze the size of the PCR product, the PCR products of the four positive samples and the genomic DNA of B31 of Borrelia burgdorferi (such as rre/M h cut/oz/en) B31 (ATCC 35210D) could not show significant differences. The four PCR-positive samples sometimes have a negative reaction in different instant quantitative pCRs, but there is a problem of poor reproducibility. This may be because 1359196 may be due to the poor design of the set of primer pairs. The sensitivity of the real-time quantitative PCR amplification reaction. In addition, when the above formula is used for conversion, the bacterial counts of the four PCR positive samples are between about 3 and 203 copies//iL, and the average bacteria 5 quantity system is 83±85 complex Ben /μί. In addition, 'selected 15 cases of clinical sensation of clinical rash with erythema erythema, suspected migratory erythema of skin rash, but serum samples were confirmed as ^ ^ ^ and IgG negative reaction cases, instant quantification Among the pcr tests, 6 of them were positive for PCR, and the positive rate was 40%. (The PCR 10 products of all positive samples were also confirmed by melting point analysis and agarose gel electrophoresis analysis. The analyzed bacteria content of all positive samples. The line is between 2 and 285 copies/pL, and the average bacterial count is 71±72 copies/pL. B. The effect of primers on the clinical detection of TWLYM-1 and TWLYM-2 in Lyme disease 15 Selected 43 Western Ink dot analysis confirmed igM positive Lyme disease

Case serum samples were subjected to real-time quantitative PCR, and the results showed that 41 PCR positive reactions were positive for 95% (Table 1). The p CR product of the 41 positive samples was further confirmed by melting point analysis and agarose gel electrophoresis analysis, wherein the average melting temperature of the PCR products of the 20 41 positive samples was 82.36±0.0786 C as determined by melting point analysis. '82.42 ° C measured with respect to the PCR product of B31 genomic DNA (ATCC 35210D) relative to B. burgdorferi Zjwrgifor/en. When the size of the PCR product was analyzed by agarose gel electrophoresis analysis, the PCR products of the 41 positive samples and the genomic DNA of Borrelia burgdorferi (such as rre/k 42 6Mrgrf〇r/m) B31 (ATCC 35210D) There is no obvious difference. Further, when the 41 positive samples were repeatedly subjected to real-time quantitative PCR using the primers of the present invention against TWLYM-1 and TWLYM-2, sensitivity and reproducibility were both high. In addition, when converted according to the formula described above, the bacterial content of the 41 positive samples is between about 27 and 3210 copies/pL, and the average bacterial amount is 359±554 copies//iL (including The amount of bacteria is 0, which is not included in the average). Further, when the serum samples of the second group of Lyme disease cases were detected by the primers of the present invention against TWLYM-1 and TWLYM-2, 4 of the 5 samples showed a positive reaction with a positive probability of 80%. The bacterial count of the four positive samples was calculated to be between about 59 and 111 replicates/pL, and the average bacterial count was 83 ± 27 replicates/μί. When the serum samples of the third group of Lyme disease cases were detected by the primers of the present invention against TWLYM-1 and TWLYM-2, 9 of the 14 samples showed a positive reaction with a positive rate of 64.2%. The bacterial count of the nine positive samples was calculated to be between about 104 and 202 replicates/μί, and the average bacterial count was 148 ± 33 replicates//iL. When the serum samples of the fourth group of Lyme disease cases were tested by TWLYM-1 and TWLYM-2, 19 of the 25 samples showed a positive reaction with a positive rate of 76% (see Table 1). The PCR products of the 19 positive samples were further confirmed by melting point analysis and agarose gel electrophoresis analysis. In addition, the bacterial count of the 19 positive samples was calculated from about 7〇 to 1143 replicates/μί′ and the average bacterial count was about 357±312 replicates//xL» 1359196 Table 1. Introduction to P1 and P2 and primer pair TWLYM-1 and TWLYM-2 for real-time quantitative PCR test results. The average bacterial rate of positive serum for Lyme disease (X soil SD) copy/μΐ bacterial amount range copy/μΐ P1 and P2 Group 1 (11) 36.3% (4/11) 83 ± 85 3^203 TWLYM-1 and TWLYM-2 Groups 1 to 3 (62) Group 1 (43) 87.1% (54/62) 95.3% (41/43) 359±554 27-3210 Group 2 (5) 80% (4/5) 83+27 59-111 Group 3 (14) 64.2% (9/14) 148±33 104~202 P1 With P2, the fourth group 40% (6/15) 71±72 2-285 TWLYM-1 and the fourth group 76% (19/25) 357±312 70~1143 TWLYM-2

All documents and patents cited in this specification are incorporated herein by reference in their entirety. In the event of a conflict, the detailed 5 description of the case (including definitions) will prevail.

While the invention has been described with reference to the specific embodiments of the present invention, many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only by the scope of the appended claims. 10 44

Claims (1)

1359196 Patent Application No. 094116064 Replacement Page :January 101101曰 10 20 X. Patent application scope: 1. A primer pair for detecting the pathogenic strain of Borrelia burgdorferi ftwrgi/or/eri sensu lato), which contains 16S according to the genus Borrelia species j/7. The rRNA gene is designed as a forward primer and a reverse primer, which is composed of a nucleotide sequence as shown in sequence identification number: 1, and the reverse primer is identified by a sequence The nucleotide sequence shown in No. 2 is composed. 2. A primer pair as claimed in claim 1 which is useful for detecting a genetic subtype comprising one of the pathogenic strains of Borrelia burgdorferi 6wrg<ior/erz·sensu lato) selected from the group consisting of Biological samples: one of the gene strains of Borrelia burgdorferi (5. sensu stricto), Borrelia burgdorferi (5., Borrelia oxysporum (5. q/ke/")) and Borrelia sinensis (5.y'a/jom'ca) 3. A test kit for detecting a pathogenic strain of Borrelia burgdorferi (5〇n*e/k sensu lato), which contains a set of a 16S rRNA gene of the Borrelia bacillus pathogenic strain CSorre/ifl sensu lato) designed to be a pair of primers comprising a forward primer and a reverse primer, the forward primer being numbered by The nucleotide sequence shown in Fig. 1 is composed of a nucleotide sequence as shown in SEQ ID NO: 2. 4. The test kit of claim 3, further comprising a nucleic acid dye for monitoring the PCR product. 5. For the inspection kit of claim 4, wherein the nucleic acid dye is S 1 1359196 10 15 094116064 Patent Application Specification Replacement Page Revision Date: January 101 曰 selected from the group consisting of: ethidium bromide (EtBr), type I blue-green nucleic acid dye (SYBR GREEN I), Type II blue-green nucleic acid dye (SYBR GREEN II), clear color nucleic acid dye (SYBR 〇range), gold nucleic acid dye (SYBR Gold), propidium iodide (pr〇pidium Iodide), blue nucleic acid dye ( SYTOX Blue), SYPRO Ruby 〇6. The test kit of claim 5, wherein the nucleic acid dye is a type I blue-green nucleic acid dye (SYBRGREENI). 7. A method for detecting in vitro whether a body has Lyme disease, comprising: for a biological sample taken from an individual suspected of having Lyme disease, using a set of patent applications The primer pair of the third term is subjected to a polymerase chain reaction, and the 16S rRNA gene of the Borrelia burgdorferi pathogenic strain sensu lato) is detected in the sample, and the Borrelia burgdorferi pathogenic strain sensu The presence of the 16S rRNA gene of iat〇) is a characterization of Lyme disease. D 8. The method of claim 7, wherein the biological sample contains a pathogenic strain of Borrelia burgdorferi CBwre/k Wrgdor /eW sensu lato) One of the gene subtypes selected from the following group: Borrelia burgdorferi gene strain (and hrgi/or/en·sensu stricto), B. burgdorferi (and, Borrelia sinensis) Bacteria (and q/ze/ii) and sputum boringobacteria (method of item 7 of the patent scope of claim 4) wherein the biological sample is selected from the group consisting of blood, A Clear, blood II, vesicle fluid, brain 20 Revision date: 101 years 1 The method of claim 9, wherein the biological sample is serum. The method of claim 9, wherein the biological method is the method of claim 9, wherein the biological method is the method of claim 9, wherein the biological method is The sample is a vesicle fluid. 12. The method of claim 7, wherein the 16S rRNA gene of the Borrelia burgdorferi pathogenic strain (5orre"a sensu lato) is obtained by using the following methodology At least one of the tests: real time quantitative PCR, nested PCR, multiplex PCR polymerase chain reaction - multiplex PCR polymerase chain reaction-single Strand conformation polymorphism, restriction fragment length polymorphism nucleic acid sequence-based amplification (NASBA) and transcription-mediated amplification (TMA) 13. The method of applying for patent item n, wherein the snail 16S rRNA genes exist body Pathogenic strains sensu lato) is by quantitative PCR using real time to be detected. The method of claim 13, wherein the total genomic DNA is extracted from the biological sample as a template for PCR prior to performing immediate quantitative PCR. 1359196 Patent Application No. 094,116,064, the entire disclosure of which is incorporated herein by reference. The nucleic acid dyes in the group were used for monitoring the PCR products: ethidium bromide (EtBr), type I blue-green nucleic acid dye (SYBR GREEN I), type II blue-green nucleic acid dye (SYBR GREEN II), SYBR orange, SYBR Gold, Propidium Iodide, blue nucleic acid dye (SYTOX Blue), sapphire nucleic acid dye (SYPRO Ruby). 16. The method of claim 15, wherein in the real-time quantitative PCR performed, the nucleic acid dye used to monitor the PCR product is a type I blue-green nucleic acid dye (SYBR GREEN I).