PCR-RLB技术同时检测淋病奈瑟菌Opa和16S rRNA基因的研究

目的建立和评价一个新的多重PCR-反向线点杂交技术(RLB)检测淋病奈瑟菌(Neisseria gonorrhoeae,NG)的方法。方法选择NG Opa基因和16S rRNA基因分别设计两对PCR引物,生物素标记下游引物。构建二重PCR扩增NG DNA,然后与固定在尼龙膜上的特异性寡核苷核探针杂交。并对117例经荧光定量PCR(FQ-PCR)检测的标本进行检测。结果多重PCR两对引物均可扩增3株NG标准菌株DNA,其中Opa和16S rRNA PCR产物的片段长度分别为89bp和414bp。43份FQ-PCR NG阳性标本中31份16S rRNA PCR-RLB阳性,而Opa PCR-RLB均检测为阳性,31份二者均为阳性。74份FQ-PCR NG阴性标本中64例Opa PCR-RLB阴性,73例16S rRNA PCR-RLB阴性,64份二者同时为阴性。结论多重PCR-RLB检测NG是一种简便、快速及有效的方法,为无症状人群NG感染的初筛和准确诊断提供了一种可靠的方法。

The Study on PCR-reverse Line Hybridization Assay for Detection Opa and 16S rRNA Gene of Neisseria Gonorrhoeae

Objective To study and evaluate a new multiplex PCR-based reverse line blot hybridization assay(mPCR-RLB) for detection of N.gonorrhoeae.Methods The DNA from N.gonorrhoeae was amplified with the two sets of primers directed to the Opa and 16S rRNA gene.The reverse primers were labeled with biotin.The products were identified by hybridization with its specific oligonucleotide probes labeled with amidogen in a Nylon membrane.The mPCR-RLB was used to detect 117 specimens that had tested by fluorescence quantify PCR(FQ-PCR).Results All 3 species of N.gonorrhoeae strains were amplified by mPCR-RLB,and the length of Opa and 16S rRNA PCR products was 89 bp and 414 bp.43 and 31 of 43 FQ-PCR positive specimens were positive by Opa and 16S rRNA PCR-RLB,31 of them were positive by both Opa and 16S rRNA PCR-RLB.64 and 73 of 74 FQ-PCR-negative specimens were negative by Opa and 16S rRNA PCR-RLB,64 of them were negative by both Opa and 16S rRNA PCR-RLB.Conclusion The mPCR-RLB is a simple,fast and effective method to the screening of asymptomatic populations for N.gonorrhoeae infections and confirmation.