HIV-1结构基因多重巢式RT-PCR方法检测

Simultenous detection of HIV-1 genes with multi-nested RT-PCR assay

目的建立一种简便、可靠的可用于诊断人类免疫缺陷病毒1型(HIV-1)感染的多重巢式逆转录-聚合酶链反应(RT-PCR)方法。方法针对广西壮族自治区HIV-1主要流行毒株pol、gag和env基因区设计PCR引物,建立同时扩增3个基因的多重巢式RT-PCR方法,检测118例HIV-1阳性样本和48例HIV-1阴性样本,并与免疫印迹法检测结果比较,对多重巢式PCR方法进行评价。结果多重巢式RT-PCR方法的检测下限为250 copies/mL,灵敏度和特异度分别为96.6%,97.9%,重复性为98.3%,与免疫印迹法的一致性为97.6%。结论本研究建立的多重巢式RT-PCR检测方法具有高灵敏度、高特异度、重复性好、快速等优点,可作为一种快速、简便、可靠的HIV-1感染辅助诊断方法。

Objective To develop an easy and reliable multi-nested RT-PCR assay for diagnosis of HIV-1 infection.Methods Several sets of PCR primers were designed to develop a multi-nested RT-PCR assay for amplification of gag,pol,and env gene of HIV-1 variants prevailing in Guangxi.Totally 118 HIV-positive samples and 48 HIV-negative samples were detected with the multi-nested RT-PCR assay and the detection results were compared to that of Western blot.Results The lowest limit of the multi-nested RT-PCR assay was 250 copies/mL and the sensitivity,specificity,and repeatability was 96.6%,97.9%,and 98.3%,respectively.The consistency between the multi-nested RT-PCR assay and Western blot assay was 97.6%.Conclusion A multi-nested RT-PCR assay with high sensitivity,specificity,repeatability,and time-saving was developed and could be applied for auxiliary diagnosis of HIV-1 infection.