多重PCR结合反向线点杂交方法检测七种性病病原体

Development of a multiple PCR-based reverse line blot hybridization assay （InPCR-RLB） to detect seven sexually transmitted disease pathogens

目的建立多重PCR结合反向线点杂交技术同时检测淋球菌、沙眼衣原体、生殖支原体、人型支原体、微小脲原体、解脲脲原体和毛滴虫的方法。方法设计7对生物素标记的上述病原体特异性引物同时扩增病原体靶基因。扩增产物与预先标记在尼龙膜上的特异性探针杂交、显影，判读结果。多重PCR结合反向线点杂交方法检测211个尿道、宫颈分泌物临床标本（男107例，女104例），并与传统单一引物PCR检测结果比较，评价反向线点杂交的敏感性和特异性。结果多重PCR结合反向线点杂交技术能敏感和特异地检测所有这7种病原体标准株，能敏感地检测病原体100拷贝的靶基因。在211个临床标本的检测中，2．8％（6／211）的标本使用多重PCR结合反向线点杂交技术检测为阳性，而单一引物PCR检测为阴性，再使用巢式PCR检测这6个标本，结果为阳性。结论多重PCR结合反向线点杂交技术能快速、敏感和特异地检测多种性病病原体。

Objective To develop a multiple PCR-based reverse line blot hydrization assay （mPCR-RLB） to simutaneously detect several STD pathogens： Neisserria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, U. parvura, Mycoplasma genitalium, M. hominis and Trichomonas vaginalis. Methods Seven pairs of biotin-labelled primer were designed and synthesized to target the 16S rRNA-23S rRNA intergenic spacer regions of Neisserria gonorrhoeae, Chlamydia trachomatis, Mycoplasma, and repetitive DNA sequence of Trichomonas to identify and subtype these pathogens. DNA was extracted from the referrence strains of seven pathogens and used as templates, mPCR was performed to simutaneously amplify the target regions of these pathogens. Then, the biotin-labelled amplicons were hybridized with membranebound specific oligonucleotide probes followed by the detection of bound amplicons with chemiluminescence assay. Serially diluted plasmids containing the target genes of pathogens were amplified with this method to detect its sensitivity. Two-hundred and eleven specimens, including 104 male urethral swabs and 107 female cervical swabs, were collected from the STD clinic of Wuhan First Hospital; mPCR-RLB and single-primer PCR were performed. For specimens with inconsistent results, nested PCR was performed to confirm the results. Results The assay sensitively and specifically identified referrence strains of the tested pathogens. The detection limit of mPCR-RLB was 100 copies for all the pathogens. Of the 211 clinical specimens, 2.8% （6/21） were negative for single-primer PCR, but positive for mPCR-RLB, and nested-PCR results were consistent with those of mPCR-RLB. Conclusion mPCR-RLB is a sensitive, specific and rapid method for the detection of STD pathogens from clinical specimens.