Study On Isolating Culture Of Chlamydia Trachomatis From Clinical Strains And Its Genotyping By PCR-RFLP

Chlamydia trachomatis (Ct) is one of the most common venereal diseasepathogen internal and abroad. It is also the main pathogen of NGU. According to theestimation of WHO, there are 93 million new cases every year and making severeeconomic burden to the society. The incidence of urogenital Ct infections has anincreasing tendency year by year and it has been a great problem jeopardizing publichealth. Urogenital Ct infection can cause many diseases .Except NGU, it also causesmany complications. At present it is thought that Ct's pathogenicity had relation withdifferent Ct types. Till date, 19 Ct serovars have been discovered. Serotyping andgenotyping the two commonly used Ct typing methods. In the two systems of Cttyping, serotyping is elaborate, time consuming and it needs high concentrationantigen which has to be obtained by passage culture. So that restricts serotypingapplication. However, genotyping can get the result in a short time, and it cansubstitute serotyping to become the main method in Ct typing. Ct genotyping is thefoundation of detecting specific Ct type's prevalence, assess treatment andre-infection in the clinic, epidemiological investigation and so on. It plays animportant role on the construction of relation between different types and differentclinical courses, deducing source, judging the prognosis. Ct genotyping mainly baseson MOMP gene (i.e. ompl gene).So it can provides beneficial information for theresearch of ompl hereditary variation mechanism, so it will facilitate the developmentof Ct vaccine.Objective: Isolating the Ct clinical strains by cell culture and PCR. Genotypingthe positive clinical strains by PCR-RFLP to understand the genotypes' distribution of STD clinic in Tianjin and research the relationship of genotype and thepatients'clinical course. Establish a maturated feasible screening culture method forclinical Ct which is suitable for our laboratory. Reserve the Ct positive clinical strainsand it will lay a foundation for further research.Methods: Collecting 213 urethral or cervix swabs from the patients who came toTianjin sexually transmitted disease institute between April to December, 2005. All ofthe patients were fit to the collection standard and we recorded their clinical relateddata. We undertook cell culture of clinical samples on the foundation of Ct E standardstrain. Then we amplified the positive samples by PCR to obtain ompl gene.Meanwhile we used plasmid PCR to screen positive clinical samples, and amplifiedthe ompl gene. And then using endonuclease Aluâ… and Mspâ… to cut the ompl gene,after 10ï¼…polyacrylamide gel verticale electrophoresis, we concluded the genotype bycomparing the results with Ct standard strains' cleavage map and summarized thedependability of Ct genotypes and patients' clinical courses and clinical features.Results: There were 27 culture positive clinical samples. After plasmid PCR andompl-PCR, we obtained 33 positive clinical strains which can be genotyped by RFLPin all. After genotyping, there were 5 genotypes, including genotype E 25,F 2,D 1,G 3 and J 2. Comparing genotype E and other types, there was a statisticalsignificance difference on the distribution of different year phase, but no statisticalsignificance difference could be found on the sex, course of disease, whether there aresexual contacts outside marriage, educational background and so on.Conclusions: 1,Genotyped Ct clinical samples from STD clinics in ourdepartment by PCR-RFLP successfully. 2,E genotype is the main Ct type in our STDclinics, this is basically accordance with reports from other regions internal andabroad, but it also needs to enlarge the sample size to get a precise ratio. 3,We gropea method suitable for clinical Ct strains cell culture, and reserved the positive strainsand lay foundations for further researches such as immunological mechanism, pathogenesis, drug sensitivities test, animal model, conservation vaccine and so on.