RU2443782C1 - Method for chlamydia trachomatis genotyping - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves DNA recovery from a clinical specimen followed by the PCR procedure with using group-specific primers to ompA Chlamydia trachomatis gene sites; the electrophoresis analysis of the amplification products is used to type Chlamydia trachomatis by B, C genotype groups and an intermediate group that is followed by the PCR procedure with using a number of type-specific primers selected by the Chlamydia trachomatis genotype group typing values; and the electrophoresis analysis of the amplification products is used to type Chlamydia trachomatis by A, B, C, D, E, F, G, J, Ja, H and K genotypes.

EFFECT: invention provides the information value and simplification of Chlamydia trachomatis typing in the infection caused by any Chlamydia trachomatis genotype combinations.

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Description

The invention relates to medicine and biology and can be used for genotyping of Chlamydia trachomatis.

Chlamydia is a widespread group of intracellular obligate parasites that cause diseases in humans and animals. Among the causative agents of chlamydia, at present, of particular importance for medicine is Chlamydia trachomatis, which causes various infectious diseases associated with a particular serotype or a similar chlamydial genotype. There are 19 serotypes of Chlamydia trachomatis, combined in three groups, which were determined using polyclonal and monoclonal antibodies to the outer main membrane protein MOMP: group B (serotypes: B, Ba, D, Da, E, LI, L2, L2a), group C (serotypes: A, C, H, I, Ja, J, K, and L3), and an intermediate group (serotypes: F, G, Ga) (Molano M., Chris JLM, Meijer SA [et al.]. Combination of PCR targeting the VD2 of ompl and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrape specimens. // Journal of clinical microbiology. - 2004. - Vol. 42, No. 7. - P.2935-2939). The method of serological typing using specific antibodies is laborious to perform in a clinical laboratory and is expensive. Currently, typing of Chlamydia trachomatis is aimed at the development and application of molecular genetic methods using variable regions of the ompA gene. Therefore, strains of Chlamydia trachomatis typed using molecular genetic methods are called genotypes (Huang C.-T., Wong W.-W., Li L.-H., Chiang C.-C, Chen B.-D., and Li S.-Y. Genotyping of Chlamydia trachomatis by Microsphere Suspension Array. // Journal of clinical microbiology. - 2008. - Vol. 46, No. 3. - P.1126-1128). Based on the pathogenetic potential, serotypes A, B, Ba, and C are associated with classical endemic trachoma; serotypes from D to K cause urogenital pathology - epididymitis, cervicitis, salpingitis, pathology of pregnancy and childbirth; serotypes L1 to L3 are causative agents of venereal lymphogranulomas (Yuan Y., Zhang YX, Watkins NG and Caldwell HD Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars. // Infection and immunity. -1989. - Vol. 57, N 4. - P.1040-1049). In addition, based on published data, serotypes G, I and D are associated with the occurrence of cervical cell carcinoma, and chronic infections caused by serotype K in women lead to infertility (Anttila T., Saikku R., Koskela P. [et al.] Serotypes of Chlamydia trachomatis and risk for development of cervical squamous cell carcinoma. // JAMA. - 2001. - Vol.285, No. 1. - P.47-51).

For epidemiological surveillance of the causative agent of chlamydial infection, to identify pathogenetic potential depending on the type of Chlamydia trachomatis, and, as a result, to ensure the effectiveness of treatment, it is necessary to monitor the circulation of types of Chlamydia trachomatis.

In clinical practice, the incidence of chlamydial infection caused by two serotypes of Chlamydia trachomatis reaches 50%. A frequent combination of serotypes D and E, E and F, K and H is detected (Molano M., Chris JLM, Meijer SA [et al]. Combination of PCR targeting the VD2 of omp1 and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrape specimens. // Journal of clinical microbiology. - 2004. - Vol. 42, N 7. - P. 2935-2939).

There is a method of detecting the LI, LII and LIII genotypes of Chlamydia trachomatis that can cause venereal lymphogranuloma based on the initial amplification of the cytotoxin gene and further sequencing of this gene, which is inherent only to these chlamydia genotypes (US patent No. 2007/0269810 A1).

The main disadvantage of this method is the inability to carry out genotyping of other genotypes of Chlamydia trachomatis, often found in human populations.

A known method for the genotyping of Chlamydia trachomatis using multilocus sequencing-typing of variable regions of the omA gene.

According to this known method, PCR was initially performed separately for each of the variable regions of the omA gene using primers:

Then, the sequencing of amplified regions of the ompA gene and further processing of the obtained sequencing data were performed using the BioEdit (7.0) and SeqScape (2.5) programs, which provide analysis of nucleotide sequences in comparison with known sequences from electronic databases (Klint M., Fuxelius H .-N., Goldkuhl RR, Skarin N., Rutemark C, Andersson SGE, Persson K., Herrmann B. High-Resolution Genotyping of Chlamydia trachomatis Strains by Multilocus Sequence Analysis. // Journal of clinical microbiology. - 2007. - Vol. .45, N 5. - P.1410-1414).

There is also a known method for determining the Chlamydia trachomatis genotype by analysis of restriction fragment length polymorphism - RFLP analysis (Restriction Fragment Length Polymorphism - RFLP analysis), based on the polymerase chain reaction (PCR) of variable regions of the ompA gene that encodes the main membrane protein of the MOMP chlamydia, and cleavage sites for fragments specific for each genotype by restriction enzymes (Rodriguez P., Vekris A., Barbey-rac B., Dutilh B., Bonnet J., and Bebear C. Typing of Chlamydia trachomatis by restriction endonuclease analysis of the amplified major outer membrane protein gene. // Journal of clinical microbiology. - 1991. - Vol.29, N 6. - P.1132-1136; Gao X., Chen XS, Yin YP, Zhong MY, Shi MQ, Wei WH, Chen Q., Peeling RW, Mabey D. Distribution study of Chlamydia trachomatis serovars among high-risk women in China performed using PCR-restriction fragment length polymorphism genotyping. // Journal of clinical microbiology. - 2007. - Vol.45, N 4. -P.1185-1189).

The main disadvantage of these known methods for determining the genotypes of Chlamydia trachomatis is their lack of information in cases where chlamydial infection is caused by several pathogen genotypes, because it is not possible to determine the specific genotypes of Chlamydia trachomatis by the length of the restriction fragments.

The prototype of the proposed method for genotyping Chlamydia trachomatis is the use of genotype typing combinations of variable regions of the ompA gene and the method of blot hybridization using type-specific oligonucleotide probes, where each probe corresponds to a specific genotype (Molano M., Chris JLM. Meijer, Morre SA, Pol R R ., and Adriaan JC van den Brule. Combination of PCR targeting the VD2 of ompl and reverse line blot analysis for typing of urogenital Chlamydia trachomatis serovars in cervical scrape specimens. // Journal of clinical microbiology. - 2004. - Vol. 42, N 7. - P.2935-2939).

The main disadvantage of the known prototype is the use of two different research methods, which complicates the genotyping and increases the number of reagents and equipment used through the use of reagents and equipment for blot hybridization.

The basis of the invention is the task of ensuring the information content of the genotyping of Chlamydia trachomatis in case of infection caused by any combination of the Chlamydia trachomatis genotypes, while simplifying the genotyping.

The problem is solved in that after DNA extraction from the clinical material, PCR is carried out using group-specific primers:

Our studies have allowed for the first time to develop primers and their combinations, providing genotyping of all types of Chlamydia trachomatis - pathogens of chlamydia and their groups. In addition, these studies proved the specificity of each of the claimed primer combinations for only one Chlamydia trachomatis genotype or one group of genotypes, which makes it possible to genotype Chlamydia trachomatis for infection caused by any two or more Chlamydia trachomatis genotypes, while simplifying the genotyping of these types of Chlamydia trachomatis.

The inventive method of genotyping Chlamydia trachomatis is new and is not described in the literature.

The technical result of the claimed invention is to provide information and simplification of genotyping of Chlamydia trachomatis in case of infection caused by any combination of Chlamydia trachomatis genotypes, while simplifying genotyping.

The invention is illustrated by the following examples, when using the proposed method of genotyping, the informativeness of the genotyping of Chlamydia trachomatis with infection caused by combinations of genotypes of Chlamydia trachomatis, while simplifying the genotyping. Moreover, the primer combinations and genotypes of Chlamydia trachomatis shown in the examples show a specific implementation of the claimed invention, but do not limit the scope of claims of the claims of the claimed invention.

Example 1. Patient S., age 9 years. The clinical diagnosis is conjunctivitis of the left eye.

After DNA extraction from scraping material from the conjunctiva of the patient, PCR was performed using group-specific primers:

Then, based on electrophoretic analysis of amplification products, which revealed a positive PCR with primers:

typed Chlamydia trachomatis groups of genotypes B and C in their combination.

Based on the typing results of the Chlamydia trachomatis genotype groups, PCR was performed using type-specific primers for genotypes E, D, H, J and R:

Then, based on electrophoretic analysis of amplification products, which revealed a positive PCR with primers:

typed Chlamydia trachomatis of genotypes E and J in their combination.

To verify the reliability and specificity of the obtained results, the amplification products of the ompA gene region of genotypes E and J were sequenced. As a result of the obtained sequencing data and their comparison with the nucleotide sequence of genotype E and J presented in the NCBI electronic database - GenBank Overview (URL: http: //www.ncbi.nlm.nih.gov/genbank/), it was found that the sequenced nucleotide sequence belongs to genotype E and genotype J.

Based on the study, the examined patient was diagnosed with chlamydial conjunctivitis caused by a combination of Chlamydia trachomatis genotypes: genotype E and genotype J.

Thus, when using the proposed method of genotyping, the informativeness of the genotyping of Chlamydia trachomatis was shown in case of infection caused by a combination of the genotypes of Chlamydia trachomatis, while simplifying genotyping that excludes the use of blot hybridization.

Example 2. Patient A., age 28 years. I complained about the absence of pregnancy. The clinical diagnosis is infertility.

After DNA extraction from scraping material from the conjunctiva of the patient, PCR was performed using group-specific primers:

Then, based on electrophoretic analysis of amplification products, which revealed a positive PCR with primers:

typed Chlamydia trachomatis group of genotypes C and the intermediate group in their combination.

Based on the results of typing the groups of Chlamydia trachomatis genotypes, PCR was performed using type-specific primers for genotypes F, G, H, Ja, J, and K:

Then, based on electrophoretic analysis of amplification products, which revealed a positive PCR with primers:

typed Chlamydia trachomatis of genotypes G and K in their combination.

To verify the reliability and specificity of the results obtained, the amplification products of the ompA gene region of genotypes G and K were sequenced. As a result of sequencing data and their comparison with the nucleotide sequences of genotypes G and K presented in the NCBI electronic database - GenBank Overview, it was found that sequenced nucleotide sequences belong to genotype G and genotype K.

The genetic typing of Chlamydia trachomatis by RFLP and a comparative analysis of the lengths of restriction DNA fragments with those presented in the NCBI - GenBank Overview electronic database specific to each genotype made it possible to determine that Chlamydia trachomatis was close to genotype G in terms of RFL profiles, however, the presence of other DNA fragments indicated the presence of another genotype, which did not allow to accurately determine the genotype as a result of the uninformativeness of the RFLP method for chlamydial infection caused by several Chlamydia trachomatis genotypes.

Based on a laboratory study, the examined patient was diagnosed with a chlamydial infection caused by a combination of the Chlamydia trachomatis genotypes: genotype G and genotype K.

Thus, when using the proposed method of genotyping, the informativeness of the genotyping of Chlamydia trachomatis was shown in case of infection caused by a combination of the genotypes of Chlamydia trachomatis, while simplifying genotyping that excludes the use of blot hybridization.

Claims (1)

The method of genotyping Chlamydia trachomatis, including the extraction of DNA from clinical material, PCR using primers to the ompA gene sections of Chlamydia trachomatis, typing of Chlamydia trachomatis by electrophoretic analysis of amplification products, characterized in that after isolation of DNA from the clinical material, PCR is performed using group-specific:
BGrl f 5′-GCTTAGATCAATCTGTTGTTGA-3 ′,
BGrl r 5′-TCTAGGCTTATGGATAGTAAAC-3 ′,
CGr f 5′-AACTTAGTTGGATTATTCGGAA-3 ′,
CGr r 5′-TCTGTATAAAGCTCAACCA-3 ′,
FI 5′-CTGTGGTGGAACTGTATACA-3 ′ and
RI 5′-TGCCACTCATGGTAATCAA-3 ′,
and then on the basis of electrophoretic analysis of amplification products typed:
Chlamydia trachomatis of genotype B group with positive PCR with primers
BGrl f 5′-GCTTAGATCAATCTGTTGTTGA-3 ′ and
BGrl r 5′-TCTAGGCTTATGGATAGTAAAC-3 ′,
Chlamydia trachomatis group of genotypes C with positive PCR with primers
CGr f 5′-AACTTAGTTGGATTATTCGGAA-3 ′ and
CGr r 5′-TCTGTATAAAGCTCAACCA-3 ′,
Chlamydia trachomatis intermediate group with positive PCR with primers
FI 5′-CTGTGGTGGAACTGTATACA-3 ′ and
RI 5′-TGCCACTCATGGTAATCAA-3 ′,
PCR is carried out using type-specific primers selected based on the typing results of a group of Chlamydia trachomatis genotypes from the series:
Comp f 5′-AAGGAAGTGTGGTCTCTGCCG-3 ′,
Comp r 5′-AATTATACAATTATTAGAACC-3 ′,
Eomp f 5′-ACACAGATACTGCCTTCTCTTG-3 ′,
Eomp r 5′-CTTGCTTGCCACTCATGGTAAT-3 ′,
Jomp f 5′-ATCTTTTTCCTAACACTGCT-3 ′,
Jomp r 5′-TTTAGGTTTAGATTGAGCAT-3 ′,
Jaomp f 5′-TACTGTCAGCGATGTAGCAG-3 ′,
Jaomp r 5′-TCCCAGATATTTAATGCCAT-3 ′,
Fh 5′-ATCTTCTGATTTTAATACAGC-3 ′,
Rh 5′-ACTTTAGGTTTAGATTGAGCA-3 ′,
Ff 5′-ACGAAACCTGCTGCAGATA-3 ′,
Rf 5′-TTGCCACTCATGGTAATCA-3 ′,
Domp f 5′-ATAATGAAAATCAAAAAAACG-3 ′,
Domp r 5′-TAGATTGAGCATATTGGAAA-3 ′,
Komp f 5′-TGTTCCTAACACTGCTTTTGGA-3 ′,
Komp r 5′-AGGTTTAGATTGAGCATATTGG-3 ′,
Gomp f 5′-AGTGTAGTCGCAGCTAAC-3 ′,
Gomp r 5′-ACTGTAACTGCGTATTTG-3 ′,
Aomp f 5′-AACACAATCTTCTGGCTTTGAT-3 ′,
Aomp r 5′-TGATTCAAAGCAGTGTTAGGA-3 ′,
Bomp f 5′-AGCCGAGACTATCTTTGATGTT-3 ′ and
Bomp r 5′-TCTGCGCTAGTTTTCACATCG-3 ′,
and then on the basis of electrophoretic analysis of amplification products typed:
Chlamydia trachomatis genotype C with positive PCR with primers
Comp f 5′-AAGGAAGTGTGGTCTCTGCCG-3 ′ and
Comp r 6′-ААТТАТАСААТТАТТАГААСС-3 ′,
Chlamydia trachomatis genotype E with positive PCR with primers
Eomp f 5′-ACACAGATACTGCCTTCTCTTG-3 ′ and
Eomp r 5′-CTTGCTTGCCACTCATGGTAAT-3 ′,
Chlamydia trachomatis genotype J with positive PCR with primers
Jomp f 5′-ATCTTTTTCCTAACACTGCT-3 ′ and
Jomp r 5′-TTTAGGTTTAGATTGAGCAT-3 ′,
Chlamydia trachomatis genotype Ja with positive PCR with primers
Jaomp f 5′-TACTGTCAGCGATGTAGCAG-3 ′ and
Jaomp r 5′-TCCCAGATATTTAATGCCAT-3 ′,
Chlamydia trachomatis genotype H with positive PCR with primers
Fh 5′-ATCTTCTGATTTTAATACAGC-3 ′ and
Rh 5′-ACTTTAGGTTTAGATTGAGCA-3 ′,
Chlamydia trachomatis genotype F with positive PCR with primers
Ff 5′-ACGAAACCTGCTGCAGATA-3 ′ and
Rf 5′-TTGCCACTCATGGTAATCA-3 ′,
Chlamydia trachomatis genotype D with positive PCR with primers
Domp f 5′-ATAATGAAAATCAAAAAAACG-3 ′ and
Domp r 5′-TAGATTGAGCATATTGGAAA-3 ′,
Chlamydia trachomatis genotype K with positive PCR with primers
Komp f 5′-TGTTCCTAACACTGCTTTTGGA-3 ′ and
Komp r 5′-AGGTTTAGATTGAGCATATTGG-3 ′,
Chlamydia trachomatis genotype G with positive PCR with primers
Gomp f 5′-AGTGTAGTCGCAGCTAAC-3 ′ and
Gomp r 5′-ACTGTAACTGCGTATTTG-3 ′,
Chlamydia trachomatis genotype A with positive PCR with primers
Aomp f 5′-AACACAATCTTCTGGCTTTGAT-3 ′ and
Aomp r 5′-TGATTCAAAGCAGTGTTAGGA-3 ′,
Chlamydia trachomatis genotype B in positive PCR with primers
Bomp f 5′-AGCCGAGACTATCTTTGATGTT-3 ′ and
Bomp r 5′-TCTGCGCTAGTTTTCACATCG-3 ′.