KR100557748B1 - Method for Identificating Borrelia burgdorferi Using PCR-RFLP - Google Patents

Abstract

The present invention relates to a method for identifying Borrelia burgdorferi , and a method for identifying and detecting Borrelia by PCR-RFLP method, wherein the groEL gene is treated with Mbo II and NIa IV restriction enzymes. It is about.

According to the present invention, Borrelia can be detected more quickly and conveniently than the conventional 16S rDNA gene comparison analysis or the RFLP method using the intergenic spacer amplicon.

Borelia, Identification, groEL, PCR-RFLP

Description

Method for Identificating Borrelia burgdorferi Using PCR-RFLP}             

1 is a photograph showing the results of electrophoresis of a restriction fragment by Mbo II and NIa IV restriction enzymes on a 10% polyacrylamide gel using the groEL gene sequence. M ₁ is 25 / 100bp Ladder, lane 1 is B. burgdorferi B31, 2 is B. bissettii DN127, 3 is B. andersonii 21123, 4 is KK2, 5 is B. afzelii VS461, 6 is B. garinii IP89, 7 is B. turdi Ya501, 8 represents B. japonica HO14, 9 represents B. valaisiana VS116, 10 represents B. lusitaniae PotiB2, 11 represents HN6, and M ₂ represents 100 bp Ladder.

The present invention relates to a method for identifying Borrelia burgdorferi , and a method for identifying and detecting Borrelia by PCR-RFLP method, wherein the groEL gene is treated with Mbo II and NIa IV restriction enzymes. It is about.

Lyme disease became known in 1905 by Swedish doctor Afzelius, but at that time it was called ACA (Acrodermatitis Chronicum Atrophicans) or Pick Herxheimer disease based on symptoms without knowing the causative organism. Later, in 1975, a number of childhood rheumatism patients in Lyme, Connecticut, USA were found to be accompanied by Erythema Chronicum Migrans, described above. It turned out that the diseases were all the same disease, which was named Lyme disease. Also it borelri the species that cause Lyme disease and relapsing fever associated Ah (Borrelia) With fungi Compare Borrelia Hallway Ferry It was named sensu lato.

Borrelia burgdorferi sensu lato is a Gram-negative microaerobic bacterium that is 20-30 µm long and 0.2-0.3 µm in diameter. Borelia, dividing by dichotomy, recursion with 7-11 flagella The number of species is 15-30. The fungus is found in the midgut of true mites (genus Ixodes ), and transmission is known to be caused by the reflux of the suspension when the tick bites.

If you bite a tick, you may experience a cold-like condition with fever, fatigue, muscle pain, joint pain, and a characteristic skin condition called Erythema migrans. If left untreated, it can cause neurological symptoms such as facial muscle paralysis, neuritis, and meningitis, as well as arthritis and myocarditis. In addition, skin symptoms such as ACA (Acrodermatitis chronica atrophicans) may occur.

Borrelia burgdorferi sensu lato has 11 known species to date. Since the causative agent of Lyme disease was isolated from ticks in 1984, a large number of Borelia corridors in countries around the world sensu lato was isolated. At first it was known as a species, but by protein staining, unicellular antibody reaction and PCR Borelri Oh corridor Perry sensu gelato the borelri Oh corridor Perry sensu stricto, borelri ah going (B. garinii), borelri ah ill jelly (B.afzelii), borelri Oh japonica (B. japonica) Etc. It was classified into four species (species).

In recent years, however, multilocus enzyme electrophoresis, DNA cleavage assays, RFLP methods using amplicons between rrf-rrl genes, and 16S rDNA sequencing have been used. B. valaisiana , B. lusitaniae , B. andersonii , B. turdi , B. tanukii , Borelli New species, such as B. bissettii and B. sinica , were added.

There have been many epidemiological studies on the relationship between species, mediators, and clinical symptoms. The Borelia corridor sensu stricto is mainly separated in the United States and Europe, while the Borelia Gani ( B. garinii ) and Borelia afzelii are separated in Europe and Asia. Traders ISO of mites spread seolka tooth (I. persulcatus) has only the strain of going borelri Oh (B. garinii) and borelri ah hurt jelly (B. afzelii). B. japonica, B. turdae and B. tanukii were separated from Japan, and Ixodes ovatus and Ixos turdus ( I. turdus ), and Iso tanuki ( I. tanuki ) are mediated ticks. Borella B. andersonii , isolated from the United States, has a mediated tick of I. dentatus . Also, B. bissettii was isolated in the United States, while B. valaisiana and B. lusitaniae were separated in Europe.

It is reported that this gene grouping may be related to the difference in clinical symptoms according to each region. For example, in the United States, the Borrelia corridor sensu stricto group is mainly isolated and has arthritis as the main symptom. In Europe, the major isolates are Borrelia gani ( B. garinii ) and Borrelia afzelii group. The neurological symptoms are mainly Borrelia gani ( B. garinii ) and skin symptoms ACA (Acrodermatitis chronica atrophicans) are associated with Borrelia afzelii ( B. afzelii ). Therefore, the development of simple classification and identification methods will enable more active epidemiological studies on the relationship between species, mediators and clinical symptoms.

In 1992, the inventors of the present invention called Ixodes nipponensis Since the first separation from mites, 23 strains were isolated from ticks and mice (Table 1). As a result of protein staining pattern, reaction pattern with unicellular antibody, and PCR method, 11 strains of 23 strains were Borelia aching. It was identified as jelly ( B. afzelii ) and 12 strains were assumed to be Borelia garniii ( B. garinii ).

However, some strains (strains isolated from Haenam, Jeonnam), which were presumed to be Borrelia stalks in unicellular antibody response, showed a different pattern from other strains in other countries. It is different from the reference strains. Therefore, it is necessary to more accurately identify whether these strains are different from the 11 known species.

Borrelia burgdorferi sensu lato isolated in Korea Separation Media Bell Test Culture water Isolate Chung-cheong bukdo mite I. nipponensis 7 KK1, KK2, KK5, CJ1, CJ2, CJ3, CJ21 rat A. agrarius 12 2 KM4, KM10 Jeollanam-do mite I. granulatus 29 4 HN6, HN7, HN8, HN9, rat A. agrarius 20 9 HN11, HN12, HN13, HN14, HN15, HN16, HN17, HN18, HN19 Gangwon-do mite I. persulcatus 16 One KW3

Borelia Hallway Ferry The identification of sensu lato has been used for various methods such as protein staining, unicellular antibody reaction, multi-locus enzyme electrophoresis, DNA cleavage assay, ribotyping, and plasmid characterization. Recently, many RFLP methods using intergenic spacer amplicons between rrf-rrl genes have been used, and 16S rDNA sequencing has been used as a gold standard.

However, the RFLP method using amplicons between rrf-rrl genes is useful for identification because the size of the target sequence is small ( 220-260 bp). In the 16S rDNA gene comparative analysis, the target sequence size (about 1,550bp) is too large to determine the sequencing.

Therefore, only a part of the hypervariable region is used for germ identification. Borelia is not easy to genetically identify only a part of the hypervariable region, and thus, Borelia must analyze a nucleotide sequence of 800 bp or more. In addition, diagnostics using species-specific primers targeting 16S rDNA have started to show problems in some species.

Therefore, the present inventors have tried to develop a new genetic classification method that can compensate for the shortcomings of the classification and identification method of Borrelia burgdorferi sensu lato ( Borlia corridor sensu lato). As a result, the GroEL protein is a chaperone. and it stored in, and the absolute need for the survival of cells and bacteria, in particular, it is known as proteins that are resistant to stress, in view of being able to reflect the evolution of borelri Ah (Borrelia) strains well, using gr oEL gene sequences It was confirmed that Borrelia strains can be identified quickly and completed the present invention.

An object of the present invention is to provide a method for identifying Borrelia strains using a PCR-RFLP method characterized in that the bacterial groEL gene is treated with Mbo II and NIa IV restriction enzymes.

Another object of the present invention is to provide a method for classifying Borrelia burgdorferi sensu lato using PCR-RFLP method, wherein the bacterial groEL gene is treated with Mbo II and NIa IV restriction enzymes. have.

Still another object of the present invention is to provide a method for detecting Borrelia strains using PCR-RFLP method.

Still another object of the present invention is to provide a method for diagnosing Lyme disease by checking whether Borrelia burgdorferi sensu lato is infected by PCR-RFLP method.

In order to achieve the above object, the present invention is a borelli using PCR-RFLP method characterized in that the groEL gene of bacteria is treated with Mbo II and NIa IV restriction enzymes to compare and confirm the size pattern of the truncated groEL gene fragment. It provides a method for the identification of fungi of Borrelia burgdorferi .

The present invention also provides a PCR-RFLP method for comparing and confirming a size pattern of a truncated groEL gene fragment by treating the groEL gene of Borrelia burgdorferi with Mbo II and NIa IV restriction enzymes. Borrelia burgdorferi sensu lato classification method is provided.

In the present invention, the size pattern of the groEL gene fragment may be characterized by comparing and confirming through an electrophoretic band, wherein the groEL gene is DNA amplified by PCR using primers of SEQ ID NO: 1 and SEQ ID NO: 2 You can do

The present invention also relates to (a) PCR using a primer pair derived from the groEL gene sequence of Borrelia burgdorferi bacteria from clinically sampled DNA from patients suspected of Borrelia burgdorferi infection. Amplifying through; And (b) treating the amplified PCR products with Mbo II and NIa IV restriction enzymes, followed by electrophoresis to identify respective specific bands. Borrelia burgdorferi bacteria It provides a detection method of.

The present invention also relates to DNA extracted from a clinical sample from a patient suspected of Borrelia burgdorferi sensu lato infection, using a primer pair derived from the groEL gene sequence of Borrelia burgdorferi . Amplifying through PCR; (b) treating the amplified PCR product with Mbo II and NIa IV restriction enzymes, followed by electrophoresis; And (c) confirming whether or not the Borrelia burgdorferi sensu lato is infected from the electrophoretic band.

In the present invention, the primer pair derived from the groEL gene sequence may be characterized in that the primer pair having a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

In particular, the following examples exemplify only a method of identifying Borrelia corridor ferri , but a method for detecting a Borreli corridor strain using a primer comprising the groEL gene sequence of the present invention and Borrelia corridor ferrissen ( Borrelia burgdorferi sensu). How to diagnose Lyme disease by confirming the infection of lato) bacteria will be apparent to those skilled in the art.

Example 1 Culture and Genomic DNA Isolation

5 ml of two isolates from Korea and 9 foreign reference strains (see Table 2) were inoculated in 100 ml of BSK-II medium and incubated at 32 ° C for 4-5 days. After incubation, the fungus sediment collected by centrifugation at 9,000 g for 10 minutes was suspended in PBS again, followed by further centrifugation at 9,000 g for 10 minutes to collect the fungal sediment. The cultured isolates were dissolved in SDS (0.5%) and proteinase K (10 μl / ml), and the fat and protein were removed using phenol-chloroform-isoamyl alcohol to obtain supernatant. The DNA was precipitated with 3M sodium acetate (pH 5.2) and ethanol, followed by centrifugation, followed by washing with 70% ethanol and drying to dissolve in TE buffer.

Strains Used in the Invention Bell Strain Source Geographic location Where to get B. burgdorferi B31 I. scapularis United States of America ATCC 35210 B. bissettii DN127 I. pacificus United States of America University of shizuoka B. andersonii 21123 I. dentatus United States of America University of shizuoka B. afzelii KK2 I. nipponensis Korea Konkuk University School of Microbiology B. afzelii VS461 I. ricinus Swiss University of shizuoka B. garinii IP89 I. persulcatus Russia University of shizuoka B. turdi Ya501 I. turdus Japan University of shizuoka B. japonica HO14 I. ovatus Japan University of shizuoka B. valaisiana VS116 I. ricinus Swiss University of shizuoka B. lusitaniae PotiB2 I. ricinus Portugal University of shizuoka Haenam strains HN6 I. granulatus Korea Konkuk University School of Microbiology

Example 2. groEL Gene sequencing

DNA extracted from the strains prepared in Example 1 as a template, GF primer (SEQ ID NO: 1, Gen. 552-572 region of GenBank Accession No. U03396 ( B. burgdorferi B31 ^T )) and GR primer (SEQ ID NO: 2, GenBank Accession No. PCR was performed using 861-841 region of U03396).

SEQ ID NO: 5'-TACGATTTCTTATGTTGAGGG-3 '

SEQ ID NO: 5'-CATTGCTTTTCGTCTATCACC-3 '

20 pmol of each primer, distilled water, and 50 ng of template DNA, the genomic DNA isolated above, were mixed to 20 μl of the final volume, and then mixed in a PCR mixture tube (AccuPower PCR PreMix, Bioneer, Korea). PCR (Perkin-Elmer Cetus, Model 9600 Thermocycler, Norwalk, USA) was performed for 5 minutes of denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 45 seconds, extension at 72 ° C for 45 seconds, and final extension at 72 ° C for 5 minutes.

The produced PCR product was cleaved by reaction with Mbo II and NIa IV restriction enzyme at 37 ° C. for 1 hour. Restriction digested fragments were identified after electrophoresis for 1 hour 10 minutes at 200V on a 10% polyacrylamide gel (FIG. 1). In Figure 1, M ₁ is 25 / 100bp Ladder, lane 1 is B. burgdorferi B31, 2 is B. bissettii DN127, 3 is B. andersonii 21123, 4 is KK2, 5 is B. afzelii VS461, 6 is B. garinii IP89, 7 represents B. turdi Ya501, 8 represents B. japonica HO14, 9 represents B. valaisiana VS116, 10 represents B. lusitaniae PotiB2, 11 represents HN6, and M ₂ represents 100 bp Ladder.

The results of FIG. 1 are summarized in Table 3. As shown in Table 3, when PCR products were cut with Mbo II and NIa IV (double cut), Borreli corridor bacteria could be classified and identified into six patterns. Burgdorferi B31 B., B. bissettii DN127, B. andersonii 21123 exhibited a 310bp fragment of B. afzelii and B. afzelii KK2 VS461 are 180, 89, showed a fragment of 41bp. B. garinii IP89, B. turdi Ya501, B. japonica HO14, B. valaisiana VS116 exhibited a fragment of each of 253, 57bp, 130, 123, 57bp, 177, 133bp, 177, 130, 3bp B. lusitaniae PotiB2 and HN6 showed fragments of 133, 98, 79, 3bp.

PCR-RFLP pattern obtained by cleaving groEL gene derived from strain used in the present invention with Mbo II and NIa IV Bell Strain Mbo II + NIa IV (double cut) GenBank Accession No. RFLP size (bp) * pattern B. burgdorferi B31 310 One U03396 B. bissettii DN127 310 One AF517974 B. andersonii 21123 310 One AF517975 B. afzelii KK2 180, 89, 41 2 AF517978 B. afzelii VS461 180, 89, 41 2 AF517954 B. garinii IP89 253, 57 3 AF517969 B. turdi Ya501 130, 123, 57 4 AF517972 B. japonica HO14 177, 133 5 AF517970 B. valaisiana VS116 177, 130, 3 5 ' AF517976 B. lusitaniae PotiB2 133, 98, 79, 3 6 AF517971 Haenam strains HN6 133, 98, 79, 3 6 AF517986

* Exact restriction fragment sizes were determined from the sequences.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention provides a method for the identification and detection of Borrelia bacteria by PCR-RFLP method characterized in that the bacterial groEL gene is treated with Mbo II and NIa IV restriction enzymes. Borrelia burgdorferi It is effective to provide a method for detecting Borrelia burgdorferi sensu lato by PCR-RFLP method characterized in that the groEL gene of bacteria is treated with Mbo II and NIa IV restriction enzymes.

According to the present invention, it is possible to identify Borrelia faster and more conveniently than the conventional 16S rDNA gene comparison analysis or the RFLP method using the intergenic space amplicon, so that diseases caused by Borrelia infection can be diagnosed quickly and accurately.

<110> Konkuk University <120> Method for Identificating Borrelia burgdorferi Using PCR-RFLP <130> KUP03-35 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer <400> 1 tacgatttct tatgttgagg g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR Primer <400> 2 cattgctttt cgtctatcac c 21

Claims (10)

The groEL gene obtained by PCR amplification of the bacterial chromosomal DNA using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 was treated with Mbo II and NIa IV restriction enzymes. Cutting; Comparing the size pattern of the truncated groEL gene fragment; And if the pattern is Pattern 1 (RFLP size 310bp), it is identified as B. burgdorferi, B. bissettii or B. andersonii , and if it is Pattern 2 (RFLP size 180,89,41bp), it is identified as B. afzelii , In case of pattern 3 (RFLP size 253,57bp), it is identified as B. garinii ; in case of pattern 4 (RFLP size 130,123,57bp), it is identified as B. turdi ; in case of pattern 5 (RFLP size 177,133bp), B is identified. In case of pattern 5 '(RFLP size 177,130,3bp), it is identified as B. valaisiana , and in case of pattern 6 (RFLP size 133,98,79,3bp), it is identified as B. lusitaniae, Haenam strains . Method of identifying Borrelia burgdorferi bacteria using a PCR-RFLP method comprising the step of. The method of claim 1 wherein the corridor borelri Oh Perry (Borrelia burgdorferi) method identified in bacteria, characterized in that is obtained through the electrophoresis patterns of the groEL gene fragment size. delete The groEL gene obtained by PCR amplification of the chromosomal DNA of Borrelia burgdorferi by PCR using primers of SEQ ID NO: 1 and SEQ ID NO: 2 was treated with Mbo II and NIa IV restriction enzymes. Cutting; Comparing the size pattern of the truncated groEL gene fragment through an electrophoretic band: and if the pattern is pattern 1 (RFLP size 310bp), it is identified as B. burgdorferi, B. bissettii or B. andersonii , and pattern 2 In case of (RFLP size 180,89,41bp), it is identified as B. afzelii , and in case of pattern 3 (RFLP size 253,57bp), it is identified as B. garinii , and in case of pattern 4 (RFLP size 130,123,57bp) In case of pattern 5 (RFLP size 177,133bp), it is identified as B. japonica , and in case of pattern 5 '(RFLP size 177,130, 3bp), it is identified as B. valaisiana , and in case of pattern 6 ( RFLP size 133,98,79,3bp) is a method for classifying Borrelia burgdorferi sensu lato using PCR-RFLP method comprising the step of identifying B. lusitaniae, Haenam strains . The method of claim 4, wherein the size pattern of the groEL gene fragment is obtained by electrophoresis. 6. The method of claim 4, wherein Borrelia burgdorferi sensu lato is characterized in that it is obtained by electrophoresis. The method of claim 4, wherein the groEL gene is DNA amplified by PCR using primers of SEQ ID NO: 1 and SEQ ID NO: 2. Borrelia burgdorferi sensu lato. delete A pair of primers for detecting Borrelia burgdorferi bacteria derived from the groEL gene sequence of Borrelia burgdorferi , and having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2. delete delete

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