两种巢式PCR方法在HIV-1早期检测的应用和比较

Application and comparison of two nested-PCRs for HIV-1 early detection

目的建立并应用HIV-1早期检测的单重巢式PCR和多重巢式PCR方法检测HIV-1感染,探讨两种方法检测结果的差异。方法针对HIV-1主要流行毒株的pol、gag、env 3个基因的保守序列设计巢式PCR引物,建立单重巢式PCR和多重巢式PCR方法。运用两种巢式PCR方法对118例HIV-1阳性样本和48例HIV-1阴性样本进行检测。结果多重巢式PCR方法的灵敏性为96.6%,高于env区单重巢式PCR的80.5%和pol区单重巢式PCR的88.1%(P<0.05),但与gag区单重巢式PCR检测结果(90.7%)差异无统计学意义(P>0.05);多重巢式PCR的特异性为99.2%,高于env区单重巢式PCR的85.4%(P<0.05),但与gag区和pol区的特异性差异无统计学意义(P<0.05);多重巢式PCR方法的平均重复性为98.3%,检测下限至少可以达到250 copy/ml。结论多重巢式PCR检测方法与单重巢式PCR检测方法相比,具有更高的灵敏性、特异性以及较好的重复性,能检测到较低拷贝数的样本,是一种快速、简便、可靠的HIV-1早期检测方法。

Objective To develop a multi-nested PCR assay for diagnosis of HIV-1 early infection. Methods Based on the consensus sequences of the gag,pol and env gene of HIV-1 variants prevailed in Guangxi, several sets of nested primers were designed to develope one single-nested PCR assay for each gene region and a multi-nested PCR assay aimed to all throe gene regions. 118 HIV-positive samples and 48 HIV-negative samples were detected by the two kinds of assay. Results The sensitivity of multi-nested PCR assay （96.6%） is higher than that of the single-nested PCR assay for the env or pol region,which is 80.5% ,88.1% respectively （P〈0.05） ,however,it does not significantly differ from that for the gag region （90.7%）. Although the specificity of the multi-nested PCR assay （99.2%） is higher than that of the single-nested PCR assay for the env region （85.4%） （P〈0.05）, there were no significant differences when compared with the gag region （93.8%） or pol region （91.7%）. The repeatability of multi-nested PCR assay is 98.3% and the lowest detection limit （LDL） can reach at least 250 copy/ml. Conclusion In compare with the single-nested PCR assay,the multi-nested PCR assay has higher sensitivity and specificity with sactisfactory repeatability and LDL. This assay is fast,simple,and reliable methods for detection of HIV-1 early infection.