反向斑点杂交技术检测泌尿生殖道沙眼衣原体及基因分型

Detection and genotyping of Chlamydia trachomatis by a reverse dot blot procedure in urogenital specimens

目的建立反向斑点杂交技术,对泌尿生殖道沙眼衣原体感染进行检测和基因分型。方法用Oligo软件设计寡核苷酸探针,采用巢式聚合酶链反应(PCR)扩增omp1基因的VS1-VS2序列,反向斑点杂交技术对沙眼衣原体进行检测和基因分型。结果巢式PCR扩增omp1基因的VS1-VS2序列的敏感性与质粒PCR符合率为98．2％(56/57)。优选出11条型特异性(A、B+Ba、C、D、E、F、G、H、I、J和K)和3条群特异性寡核苷酸探针：B群(B、Ba、D和E型),C群(A、C、H、I、J和K型)和中间群(F和G型)。特异性经过基因库中的BLAST程序比较和试验优化,结果显示各探针均与标准株呈特异性杂交。56例VS1- VS2 PER阳性的临床标本杂交法检测均阳性,共检出59株沙眼衣原体,包括8个基因型,其中以E、F、D和H型为主,占77．9％,分别为25．4％,22．0％,16．9％和13．6％。发现3例混合感染占5．4%,分别为D/E、D/F和F/K。结论反向斑点杂交技术简便且快速,可直接对临床标本沙眼衣原体进行检测和基因分型。

Objective To develop a reverse dot blot （ RDB ） technology for detecting and genotyping of urogenital Chlamydia trachomatis. Methods The oligonucleotide probes were designed by using a soft-ware （ Oligo ）. The VS1-VS2 of C. trachomatis ompl gene was amplified by a nested PCR and then the labeled amplicons were hybridized with serotype- and group-specific oligonucleotide probes for the propose of detection and genotyping. Results The concordance rate was 98.2% （ 56/57 ） when the sensitivities of nest VS1-VS2 PCR and plasmid PCR were compared in 60 clinical samples. Eleven serotype-specific probes （ A, B＋Ba, C, D, E, F, G, H, I, J and K ） and 3 group-specific probes （ group B （ B, Ba, D and E ）, group C （ A, C, H, I, J and K ） and intermediate group （ F and G ） ）, targeting the VS1-VS2 region of omp I gene of 11 different serovars of C. trachomatis, were optimized by the Nucleotide-nucleotide Blast （ blastn ） program and different reaction conditions. The probes were shown to have specific reactions with the corresponding reference strains. The 56 positive samples detected by the nested PCR all showed positive hybridization by RDB method. A total of 59 strains of C. trachomatis were identified. They included eight genotypes, and types E, F, D and H were the most common （ 77.9% ）. Multiple infections with serovars D/E, D/F, F/K, were found in 3 cases （ 5.4% ）, respectively. Conclusions RDB technology can directly detect and genotype C. trachomatis from clinical samples. It is simple, rapid and specific, and is useful for large epidemiological studies and for differentiating clinical re-infection from mixed infection.