RU2378364C1 - Method for molecular typing chlamydia trachomatis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents the method for molecular typing Chlamydia trachomatis by sequencing the variable regions of three Chlamydia trachomatis genes, such as ompA, tsf, pmpF, produced of bacterial DNA Chlamydia trachomatis recovered from a clinical material of patients suffering from urogenital Chlamydia and underwent amplification followed with restoration of analysed gene sequences, alignment and comparative analysis of nucleotide sequences.

EFFECT: possibility to improve resolution of the method for molecular typing Chlamydia trachomatis, reproducibility and efficiency of the method, to facilitate interpretation of the obtained results and ease of effect.

2 tbl, 3 ex

Description

The invention relates to microbiology and is a method of molecular typing of Chlamydia trachomatis. The invention can be used to study the epidemiology of urogenital chlamydia, assess the genetic affinity of individual strains of the causative agent of urogenital chlamydia - C.trachomatis - and identify strains of C.trachomatis that determine the nature of the clinical course of the disease.

Chlamydia as a causative agent of various human pathologies are very heterogeneous. Among more than 20 representatives of the Chlamydiaceae family, combined into two genera (Chlamydia and Chlamydophila), at least three types of chlamydia causing human diseases are known:

- Chlamydia pneumoniae (according to the modern nomenclature of Chlamydophila pneumoniae) is a causative agent of respiratory infections, the role of this microorganism in the development of atherosclerosis is shown in the experiment, but this has not been clearly proved in the clinic;

- Chlamydia psittaci (according to the modern nomenclature of Chlamydophila psittaci) - the causative agent of psittacosis;

- Chlamydia trachomatis - a causative agent of infections of the urogenital tract and several others;

- other chlamydia can cause animal diseases or lack pathogenic properties.

The causative agent of urogenital chlamydia Chlamydia trachomatis - refers to obligate pathogens and is transmitted from person to person mainly through sexual contact. In recent years, the incidence of urogenital chlamydial infection in the Russian Federation has come first among all sexually transmitted bacterial infections (STIs). In our country today, urogenital chlamydia is the second most common registered STI after trichomoniasis.

The development of effective measures to reduce the incidence of urogenital chlamydia should be based on clear ideas about the characteristics of the epidemiology of this disease. One of the main conditions for resolving these issues is the possibility of identifying the similarities and differences between bacterial strains of the same species isolated from different patients, that is, typing.

One of the criteria for choosing a typing method is the reproducibility of the method and the resolution (discriminatory) ability. At the same time, the results obtained should be easily taken into account and interpreted without involving additional expertise, which means the ability of the method to evaluate how individual types, strains, randomly selected from the bacterial population. The Simpson Diversity Index (D) [Gaston, M.A. and P.R. Hunter. 1989. Efficient selection of tests for bacteriological typing schemes. J Clin Pathol 42: 763-766; Hunter, P. R. and M. A. Gaston. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol 26: 2465-2466.], Which is calculated by the formula:

where S is the number of groups into which this method divides the entire sample of strains, n _j is the number of strains in the jth group and N is the total number of strains in the studied sample.

Ideally, for methods with maximum resolution, the Simpson index should be 1. This means that the method can be attributed to any type of any two strains.

Other selection criteria for molecular typing may include:

- the ability to analyze a large number of samples; for laboratories conducting extensive epidemiological studies, this criterion may be determining;

- the storage of genetic profiles, that is, the creation of a database, is also an important criterion for choosing a method.

The literature describes various molecular typing techniques for C.trachomatis.

Until recently, the main typing method for C. trachomatis was the serotyping method, based on the identification of variants of the antigenic structure of the main protein of the microorganism's outer membrane (MOMP). 19 C. trachomatis serovars have been described and it has been established that serotypes A, B and C cause trachoma, serotypes D to K - urogenital chlamydia and three serotypes (L1-L3) - venereal lymphogranuloma; urogenital chlamydia is most often associated with D-F serovars [Spaargaren J., Verhaest I., MooiJ S. et al. Analysis of Chlamidya trachomatis. Serovar distribution changes in the Netherlands (1986-2002). Sex Transm Infect 2004, 80: 151-152]. At the same time, serotyping is a rather laborious and lengthy process, requiring a pure chlamydia culture, highly qualified personnel and the presence of a set of monoclonal antibodies [Wang S.P., Kuo S.S., Bames R.C. et al. Immunotyping of Chlamidya trachomatis with monoclonal antibodies. J Infect Dis 1085,152: 791-800; Osserwarde J.M., Rieffe M. De Vries., Derksen-Navrocki R.P. et al. Comparison of two panels of monoclonal antibodies for determination of Chlamidya trachomatis serovars. J Clin Microbiol 1994, 32: 2968-2974]. In connection with these facts, traditional serotyping is currently being supplanted by molecular methods based on the amplification of a gene encoding the main protein of the outer membrane and subsequent analysis of the amplicon.

For amplification of C.trachomatis DNA, nested PCR is most often used. The resulting amplicons are studied by analysis of restriction fragment length polymorphism, reverse hybridization.

[Stothard, D.R. 2001. Use of a reverse dot blot procedure to identify the presence of multiple serovars in Chlamydia trachomatis urogenital infection. J Clin Microbiol 39: 2655-2659 No.; Xiong, L., F. Kong, H. Zhou, and G. L. Gilbert. 2006. Use of PCR and reverse line blot hybridization assay for rapid simultaneous detection and serovar identification of Chlamydia trachomatis. J Clin Microbiol 44: 1413-1418.]., Oligonucleotide chips [Zheng, H. P., L. F. Jiang, D.Y. Fang, Y.H. Xue, Y.A.Wu, J.M. Huang, and Z.Y.Ou. 2007. Application of an oligonucleotide array assay for rapid detecting and genotyping of Chlamydia trachomatis from urogenital specimens. Diagn Microbiol Infect Dis 57: 1-6] and sequencing [Bandea, C. I., K. Kubota, T. M. Brown, PH Kilmarx, V. Bhullar, S. Yanpaisam, P. Chaisilwattana, W. Siriwasin, and CMBlack. 2001. Typing of Chlamydia trachomatis strains from urine samples by amplification and sequencing the major outer membrane protein gene (ompi). Sex Transm Infect 77: 419-422; Klint, M., M. Lofdahl, C. Ek, A. Airell, T. Berglund, and B. Herrmann. 2006. Lymphogranuloma venereum prevalence in Sweden among men who have sex with men and characterization of Chlamydia trachomatis ompA genotypes. J Clin Microbiol 44: 4066-4071; Stothard, D.R., G. Boguslawski, and R. B. Jones. 1998. Phylogenetic analysis of the Chlamydia trachomatis major outer membrane protein and examination of potential pathogenic determinants. Infect Immun. 66: 3618-3625.]. More recently, a real-time PCR method has been proposed for typing C.trachomatis that has the same resolution as the omp1 gene sequencing (Jalal, B., N. Stephen, S. Alexander, C. Came, and C. Sonnex. 2007. Development of real-time PCR assays for genotyping of Chlamydia trachomatis. J Clin Microbiol 45: 2649-2653.]. Using some of the modern molecular typing techniques will produce identical results to traditional serotyping.

The most widely used method for molecular typing of a number of microorganisms is Multilocus Sequence Typing (MLST).

The principle of the MLST method is based on sequencing and subsequent analysis of the nucleotide sequences of a large number (from 6 to 16) of the microorganism's housekeeping genes. One of the first bacteria to be typed using the MLST method was meningococci [Feavers, I.M., S.J. Gray, R. Urwin, J.E. Russell, J.A. Bygraves, E.B. Kaczmarski, and M.C. Maiden. 1999. Multilocus sequence typing and antigen gene sequencing in the investigation of a meningococcal disease outbreak. J Clin Microbiol 37: 3883-3887].

When typing using the MLST method C.trachomatis [K-lint, M., N.N. Fuxelius, R.R. Goldkuhl, N.Skarin, C.Rutemark, S.G. Andersson, K. Persson, and B. Herrmann. 2007. High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis. J Clin Microbiol 45: 1410-1414.] Highly variable regions were used as targets for typing: two annotated genes (hctB and pbpB) and three hypothetical genes (CT058, CT144 and CT172).

The main disadvantages of this method: when reproducing the described MLST method, a number of difficulties arise associated with the selection of primers for amplification of genes and reaction conditions. In addition, the method requires the isolation of chlamydia in a pure culture. Typing on five genes is quite expensive and time-consuming.

This method is the closest to the claimed method of molecular typing of C.trachomatis. This method is selected as the closest analogue of the proposed method.

The objective of the invention is to develop a more effective method of molecular typing of C.trachomatis.

The technical result of the invention is to increase the resolution of the method of molecular typing C.trachomatis, reproducibility and productivity of the method, as well as to increase the ease of interpretation of the results and ease of implementation.

This is achieved due to the fact that from the clinical material obtained from patients with urogenital chlamydia, bacterial DNA of C.trachomatis is isolated; amplification fragments of the variable regions of the three C.trachomatis genes, such as ompA, tsf and pmpF, are obtained by polymerase chain reaction; carry out their sequencing, restoration of the sequences of the analyzed genes, alignment and comparative analysis of nucleotide sequences.

Characterization of genes selected for typing C.trachomatis:

ompA is a gene encoding the main protein of the outer membrane of C.trachomatis;

tsf — gene encoding C.trachomatis elongation factor (EF-TS); plays an important role in protein biosynthesis, and is also involved in the regulation of other C.trachomatis proteins associated with the development cycle of the microbial cell, which may be important in changing the phenotypic properties of the microorganism and determine phenotypic differences between C.trachomatis serovars [Zhang Y., Tao J ., Zhou M. et al. Elongation factor Ts of C.trachomatis: structure of the gene and properties of the protein. Arch Biochem Biophys 1997, 344: 43-52; Brunelle B. W, Nicholson T.L., Stephens R.S. Microarrey-based genomic surveying of gene polymorphisms in C.trachomatis. Genome Biology 2004, 5: R42];

pmpF is a member of the family of Chlamydiales genes located in the outer membrane and encoding polymorphic membrane proteins responsible for autotransport, translocation of the N-terminal region of the cell surface of C.trachomatis and adhesion of the microorganism to the cells of the host [Gomes J.P., Nunes A., Bruno W.J. et al. Polymorphisms in the Nine Polymorphic Membrane Proteins of C.trachomatis across All Serovars: Evidence for Serovar Da recombination and Correlation with Tissue Tropism. J Bacteriol Jan 2006: 275-286].

The method is as follows.

From the clinical material obtained from patients with urogenital chlamydia, C.trachomatis bacterial DNA is isolated in a standard way.

Amplified fragments of the variable regions of the three C.trachomatis genes (ompA, tsf, pmpF) are obtained by polymerase chain reaction. The characteristics of the target genes used for typing C.trachomatis, primers for their amplification, and the conditions for the polymerase chain reaction for C.trachomatis are presented in Table 1.

Table 1
Target genes used for typing C.trachomatis, primers for their amplification and conditions for the formulation of the polymerase chain reaction gene Primer Name 5'-3 'sequence Amplicon size bp Amplification Conditions one ompA ompAf ctaagaaaagatcctaatataa 1260 30 cycles 2 ompAr gatagcgagcacaaagaga 95 ° C - 30 sec 56 ° C - 15 sec 72 ° C - 20 sec 3 tsf tsff tccagaagacaaagtgctcaa 823 30 cycles four tsfr gagcgacttctccatggaaa 95 ° C - 30 sec 60 ° C - 15 sec 72 ° C - 20 sec 5 pmpF pmpF1f
pmpF2f
pmpF3f tatcctttgcttgcctcagtt
ggttacattgacatccgagat
cctataccatcacgcttaataa 1032 30 cycles 1190 95 ° C - 30 sec 6 pmpF1r
pmpF2r
pmpF3r caggcagcagagttatttaga
gcgatggaggtagcagatgt
gctcctcctgcattgatgta 1130 60 ° C - 15 sec 72 ° C - 20 sec

Sequencing reactions are carried out using the d-Rhodamine Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions. An analysis of the results of the sequencing reaction is carried out on an ABI-3130 instrument (Applied Biosystem, USA).

The restoration of the sequences of the analyzed genes, alignment and comparative analysis of the nucleotide sequences is carried out using the ContigExprress and AlignX modules of the Vector NTI® 9.0 software package (Informax, Inc).

The nucleotide sequences of the studied genes obtained as a result of sequencing are compared with the nucleotide sequences of the alleles of the corresponding genes in the GeneBank database using the BLAST service (http://www.ncbi.nlm.nih.gov/BLAST).

Based on the nucleotide sequence, the C.trachomatis strain is assigned a sequence type number, which is placed in a database for subsequent analysis of the clonal affinity of C.trachomatis strains.

Example No. 1

A male patient was examined (1) with suspected presence of a sexually transmitted infection. Examination of scraping material of the urethra by PCR (test system NPF "Liteh", "Polymik-Chl", Moscow) revealed C.trachomatis DNA in the patient.

C.trachomatis was typed in accordance with the proposed method.

The stages of the study included:

- isolation of total bacterial DNA of C.trachomatis from scraping material of the urethra using the DNA EXPRESS DNA isolation kit (LLC NPF Litekh, TU-9398-450-17253567-03);

- selection of primers for the polymerase chain reaction in order to identify and amplify fragments of the genes otpA, tsf and pmpF (table 1);

- carrying out a polymerase chain reaction in order to obtain amplitudes of fragments of the studied C. trachomatis genes in accordance with the reaction conditions described in table 1 and using the following reagents: 10xReaction buffer [solution: 0.66 M Tris-HCL (Cat. No. T 6791 Sigma, USA); 25 mM magnesium chloride (Cat. No. 960-9, Sigma, USA); a solution of deoxynucleotide triphosphates (DNTF) [aqueous solution of dNTP-dATP (cat. No. T3-100, NPO Vector, Novosibirsk); dGTF (cat. No. T3-PO, NPO Vektor, Novosibirsk); DCTF (cat. No. T3-130, NPO Vector, Novosibirsk); dTTF (cat. No. T3-120, NPO Vektor, Novosibirsk)]; the concentration of each dNTP - 2.5 mm]; deposited water [Millipore, USA (Cat. No. ZPMG 23004)], Taq polymerase (Promcga, USA);

- the results of amplification reactions were evaluated in a 2% agarose gel (agarose (Cat. No. A 9918, Sigma, USA), ethidium bromide solution (1% solution (Cat. No. 11615, Merk, Germany)), 50-fold TAE buffer: 2 M Tris-acetate (Cat. No. T 1258, Sigma, USA), 0.05 M Ethylenediaminetraacetic acid diatrium salt (Cat. No. 9884, Sigma, USA), pH 8.2);

- visualization of amplification products was carried out using a video system for documenting gel electrophoregrams on the appearance of luminous orange-red bands upon irradiation with UV radiation with a wavelength of 310 nm;

- formulation of the sequencing reaction using the d-Rhodamine Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions; analysis of the results of the sequencing reaction is carried out on an ABI-3130 device (Applied Biosystem, USA);

- restoration of the sequences of the analyzed genes, alignment and comparative analysis of nucleotide sequences using the ContigExprress and AlignX modules of the Vector NTI® 9.0 software package (Informax, Inc).

The nucleotide sequences of the studied genes obtained as a result of sequencing were compared with the nucleotide sequences of the alleles of the corresponding genes in the GeneBank database using the BLAST service (http://www.ncbi.nlm.nih.gov/BLAST).

As a result of sequencing of the fragment of the gene opA, the presence of the replacement of A by G at position 35 was established.

Sequencing of the tsf gene fragment revealed the substitution of C for T at position 44.

As a result of sequencing of the pmpF gene fragment, the presence of the replacement of A by C at position 134 was established.

Based on the nucleotide sequences of fragments of the ompA, tsf, and pmpF genes, the C. trachomatis strain was assigned sequence number 10; the sequence-type number is placed in the database for subsequent analysis of the clonal affinity of C.trachomatis strains.

Example No. 2

A female patient (2) was examined to exclude urogenital chlamydia, which was the patient's sexual partner (1). Examination of scraping material of the cervix and urethra by PCR (test system NPF "Litekh", "Polymik-Chl", Moscow) revealed C.trachomatis DNA in the patient.

C.trachomatis was typed in accordance with the proposed method.

The stages of the study included:

- isolation of total bacterial DNA of C.trachomatis from scraping material of the cervix and urethra using the DNA EXPRESS DNA isolation kit (OOO NPF Litekh, TU-9398-450-17253567-03);

- selection of primers for the polymerase chain reaction in order to identify and amplify fragments of the genes otpA, tsf and pmpF (table 1);

- carrying out a polymerase chain reaction in order to obtain amplitudes of fragments of the C.trachomatis genome in accordance with the reaction conditions described in table 1 and using the following reagents: 10xReaction buffer [solution: 0.66 M Tris-HCL (Cat. No. T 6791, Sigma, USA); 25 mM magnesium chloride (Cat. No. 960-9, Sigma, USA); 0.05 M ethylenediaminetraacetic acid diatrium salt (Cat. No. E 9884, Sigma, USA), pH 8.2; a solution of deoxynucleotide triphosphates (DNTF) [aqueous solution of dNTP-dATP (cat. No. T3-100, NPO Vector, Novosibirsk); dGTF (cat. No. T3-PO, NPO Vektor, Novosibirsk); DCTF (cat. No. T3-130, NPO Vector, Novosibirsk); dTTF (cat. No. T3-120, NPO Vektor, Novosibirsk)]; the concentration of each dNTP - 2.5 mm]; deposited water [Millipore, USA (Cat. No. ZPMG 23004)], Taq polymerase (Promcga, USA);

- the results of amplification reactions were evaluated in a 2% agarose gel (agarose (Cat. No. A 9918, Sigma, USA), ethidium bromide solution (1% solution (Cat. No. 11615, Merk, Germany)), 50-fold TAE buffer: 2 M Tris-acetate (Cat. No. T 1258, Sigma, USA), 0.05 M Ethylenediaminetraacetic acid diatrium salt (Cat. No. 9884, Sigma, USA), pH 8.2);

- visualization of amplification products was carried out using a video system for documenting gel electrophoregrams on the appearance of luminous orange-red bands upon irradiation with UV radiation with a wavelength of 310 nm;

- formulation of the sequencing reaction using the d-Rhodamine Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions; analysis of the results of the sequencing reaction is carried out on an ABI-3130 device (Applied Biosystem, USA);

- restoration of the sequences of the analyzed genes, alignment and comparative analysis of nucleotide sequences using the ContigExprress and AlignX modules of the Vector NTI® 9.0 software package (Informax, Inc).

The nucleotide sequences of the studied genes obtained as a result of sequencing were compared with the nucleotide sequences of the alleles of the corresponding genes in the GeneBank database using the BLAST service (http://www.ncbi.nlm.nih.gov/BLAST).

As a result of sequencing of the fragment of the gene opA, the presence of the replacement of A by G at position 35 was established.

Sequencing of the tsf gene fragment revealed the substitution of C for T at position 44.

As a result of sequencing of the pmpF gene fragment, the presence of the replacement of A by C at position 134 was established.

Based on the nucleotide sequences of fragments of the ompA, tsf, and pmpF genes, the C. trachomatis strain was assigned sequence number 10; the sequence number is placed in the database for subsequent analysis of the clonal affinity of C.trachomatis strains.

As a result of typing of C.trachomatis isolated from patients (1) and (2), who were sexual partners, the presence of both partners of C.trachomatis belonging to the same sequence type in the urogenital tract was established.

The data obtained allow us to conclude that the used C.trachomatis typing method can be used to establish infection of sexual partners (in particular, in a forensic medical examination) and to study the pathways of urogenital chlamydial infection.

Example No. 3

A female patient was examined (3) with suspected presence of a sexually transmitted infection. Examination of scraping material of the cervix and urethra by PCR (test system NPF "Litekh", "Polymik-Chl", Moscow) revealed C.trachomatis DNA in the patient.

C.trachomatis was typed in accordance with the proposed method.

The stages of the study included:

- Isolation of total bacterial DNA of C. trachomatis from scraping material of the cervix and urethra using the DNA EXPRESS DNA isolation kit (000 NPF Litekh, TU-9398-450-17253567-03);

- selection of primers for the polymerase chain reaction in order to identify and amplify fragments of the genes otpA, tsf and pmpF (table 1);

- carrying out a polymerase chain reaction in order to obtain amplitudes of fragments of the C.trachomatis genome in accordance with the reaction conditions described in table 1 and using the following reagents: 10xReaction buffer [solution: 0.66 M Tris-HCL (Cat. No. T 6791, Sigma, USA); 25 mM magnesium chloride (Cat. No. 960-9, Sigma, USA); 0.05 M ethylenediaminetraacetic acid diatrium salt (Cat. No. E 9884, Sigma, USA), pH 8.2; a solution of deoxynucleotide triphosphates (DNTF) [an aqueous solution of dNTP - dATP (cat. No. T3-100, NPO Vector, Novosibirsk); dGTF (cat. No. T3-PO, NPO Vektor, Novosibirsk); DCTF (cat. No. T3-130, NPO Vector, Novosibirsk); dTTF (cat. No. T3-120, NPO Vektor, Novosibirsk)]; the concentration of each dNTP - 2.5 mm]; deposited water [Millipore, USA (Cat. No. ZPMG 23004)], Taq polymerase (Promcga, USA);

- the results of amplification reactions were evaluated in a 2% agarose gel (agarose (Cat. No. A 9918, Sigma, USA), ethidium bromide solution (1% solution (Cat. No. 11615, Merk, Germany)), 50-fold TAE buffer: 2 M Tris-acetate (Cat. No. T 1258, Sigma, USA), 0.05 M Ethylenediaminetraacetic acid diatrium salt (Cat. No. 9884, Sigma, USA), pH 8.2);

the amplification products were visualized using a video system for documenting gel electrophoregrams on the appearance of luminous orange-red bands upon irradiation with UV radiation with a wavelength of 310 nm;

- formulation of the sequencing reaction using the d-Rhodamine Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions; analysis of the results of the sequencing reaction is carried out on the device AB 1-313 0 (Applied Biosystem, USA);

- restoration of the sequences of the analyzed genes, alignment and comparative analysis of nucleotide sequences using the ContigExprress and AlignX modules of the Vector NTI® 9.0 software package (Informax, Inc).

The nucleotide sequences of the studied genes obtained as a result of sequencing were compared with the nucleotide sequences of the alleles of the corresponding genes in the GeneBank database using the BLAST service (http://www.ncbi.nlm.nih.gov/BLAST).

As a result of sequencing of the fragment of the gene opA, the presence of the replacement of A by C at position 97 was established.

Sequencing of the tsf gene fragment revealed the substitution of C for T at position 44.

As a result of sequencing of the pmpF gene fragment, it was found that G is replaced by A at position 347.

Based on the nucleotide sequences of the fragments of the genes otpA, tsf, and pmpF, the C. trachomatis strain was assigned sequence number 12; the sequence number is placed in the database for subsequent analysis of the clonal affinity of C.trachomatis strains.

As a result of typing of C.trachomatis isolated from patients (1) and (2), who were half partners, and patient (3), it was found that in the urogenital tract of the examined sexual partners - patients (1) and (2), C. trachomatis, belonging to one sequence type, and in the urogenital tract of the patient (3) C. trachomatis, belonging to another sequence type, is present. Thus, the applied C.trachomatis typing method revealed a difference between the two C.trachomatis strains obtained from non-sexual partners, which indicates its high discriminatory ability.

The data obtained allow us to conclude that the applied C. trachomatis typing method can be used to assess the genetic affinity of individual strains of the pathogen of urogenital chlamydia, as well as the pathways of the distribution of urogenital chlamydial infection.

When comparing the claimed typing method with the other most common methods, the following advantages of this method were revealed:

- sequencing of variable regions of three C.trachomatis genes with pronounced nucleotide polymorphism allows (in comparison with genotyping for one gene) to significantly increase the resolution and reproducibility of the C.trachomatis typing method;

- the study of a small (in comparison with the MLST method) number of C.trachomatis genes can significantly reduce the cost and reduce the time for conducting studies on typing of C.trachomatis strains and increase its productivity; and also increase the ease of interpretation of the results and ease of implementation.

Comparative characteristics of the proposed and known methods of molecular typing of C.trachomatis are shown in table 2.

Claims (1)

The method of molecular typing of Chlamydia trachomatis, comprising isolating from clinical material obtained from a patient with urogenital chlamydia, bacterial DNA of Chlamydia trachomatis, obtaining amplified fragments of variable regions of the Chlamydia trachomatis genes and sequencing them with subsequent restoration of the sequences of the analyzed genes by comparative analysis, alignment and characterized in that amplified fragments of three variable regions of the Chlamydia t genes are obtained rachomatis, such as ompA, tsf, pmpF.