泌尿生殖道沙眼衣原体MLVA-ompA分型方法的建立及初步应用

Establishment and Application of MLVA-ompA Typing for Chlamydia trachomatis

目的:探讨泌尿生殖道沙眼衣原体(Ct)多位点可变数目串联重复序列分析(MLVA)-omp A分型方法的建立及初步应用。方法:共对5株Ct参考株和31株性病门诊男性Ct阳性株进行MLVAomp A分型。合成针对串联重复次数差异性显著的3个可变数目串联重复序列(VNTR)(CT 1291,CT1299,CT 1335)的引物,优化PCR反应条件,进行PCR扩增,与参考序列比对进行MLVA分型。Omp A分型采用本实验室已建立的巢式PCR方法,扩增omp A基因,通过测序后进行Blast比对判断型别。结果:omp A和MLVA分型方法的分辨率分别为0.83和0.86,MLVA-omp A分型分辨率可达0.96。31株临床Ct株中,29例(93.5%)omp A分型成功,共被分为8个基因型B(0.7%)、D(37.9%)、E(0.7%)、F(10.3%)、G(0.7%)、H(0.7%)、J(20.7%)、K(3.4%)。31株临床Ct株MLVA分型全部成功(100%),共检出10个MLVA亚型和18个MLVA-omp A亚型。结论:成功建立了Ct的MLVA-omp A分型方法,该方法可对临床Ct阳性株进行分型分析,与omp A分型方法相比,具有更高分辨率,可作为Ct传统分型方法的补充。

Objective：To establish and applicate the muhilocus variable number of tandem re- peats analysis （MLVA）-ompA method for Chlamydia trachomatis typing. Methods： Five refer- ence genotypes and 31 C. trachomatis positive specimens from men were genotyped by MLVA- ompA. Five pairs of specific primers were designed for amplifying the 3 fragments of CT 1291, CT 1299, CT1335. The reaction was optimized against temperature, concentration and reaction time. Nested PCR was performed to amplify the ompA gene. All the amplified products were sent to se- quence. The assignment of MLVA types were carried out manually according to the reference se- quences. OmpA sequences were genotyped via comparison to the NCBI database via BLAST. Re- suits： The discriminatory index D was 0. 83 for ompA, O. 86 for MLVA and 0.96 for MLVA- ompA. 93.5% of 31 specimens were genotyped by ompA. Genotyping of the ompA gene allowed clinical samples to be divided into eight genotypes： B （0.7%）, D（37.9% ）, E（0. 7% ）, F （ 10. 3% ）, G（0. 7% ）, H（0.7% ）, J （20. 7% ）, K （3.4%）. All specimens from men were di- vided into 10 MLVA and 18 MLVA-ompA genotypes. Conclusion： The MLVA-ompA genotyping method was established successfully, it can be used for genotyping of Chlamydia trachomatis, which represented a more favorable degree of discrimination than ompA and could be a comple- ment for the subtyping of Chlamydia trachomatis.