Grankvist O, Walther L, Bredberg-Rådén U, Lyamuya E, Mhalu F, Gustafsson A, Biberfeld G, Wadell G
Department of Virology, University of Umed, Sweden.
J Virol Methods. 1996 Dec;62(2):131-41. doi: 10.1016/s0166-0934(96)02094-0.
To investigate the efficacy of the SK431/SK145 primer pair and two nested primer assays in amplifying African HIV-1 samples, a total of 35 Tanzanian PBMC samples were examined. These were assayed by two HIV-1 specific nested in-house PCR assays and a commercial HIV-1 PCR kit (GeneAmp) using SK431/SK145 as the primer pair. One of the nested PCR assays has been evaluated previously (old assay), whereas the modified assay was constructed from the HIV-1 sequence alignment released in August 1993. The modified nested primer assay showed increased sensitivity in the gag and env regions compared to the old nested primer assay. However, both the old and the modified nested primer assays displayed higher sensitivity for the detection of Tanzanian HIV-1 proviruses than the GeneAmp assay. When two regions were used (gag and env) as targets for the amplification, the modified nested primer assay detected 97.1% (34/35) of the proteinase K lysed samples, compared to 68.6% (24/35) using the SK431/SK145 primer pair (P < 0.01**). The results indicate that the SK431/SK145 primer pair may be less suitable when HIV-1 samples from Africa are analysed. The results also show that continuous modification of primer sequences can improve and maintain high sensitivity for the detection of highly divergent HIV-1 strains.
为研究SK431/SK145引物对及两种巢式引物检测法扩增非洲HIV-1样本的效果，共检测了35份坦桑尼亚外周血单核细胞（PBMC）样本。采用两种HIV-1特异性巢式内部PCR检测法及一种商用HIV-1 PCR试剂盒（GeneAmp），以SK431/SK145作为引物对进行检测。其中一种巢式PCR检测法先前已进行过评估（旧检测法），而改良检测法是根据1993年8月发布的HIV-1序列比对构建的。与旧巢式引物检测法相比，改良巢式引物检测法在gag和env区域显示出更高的灵敏度。然而，旧的和改良的巢式引物检测法在检测坦桑尼亚HIV-1前病毒方面均比GeneAmp检测法具有更高的灵敏度。当使用两个区域（gag和env）作为扩增靶点时，改良巢式引物检测法检测出97.1%（34/35）的蛋白酶K裂解样本，而使用SK431/SK145引物对的检测率为68.6%（24/35）（P < 0.01**）。结果表明，分析来自非洲的HIV-1样本时，SK431/SK145引物对可能不太适用。结果还表明，引物序列的持续改良可提高并维持对高度分化的HIV-1毒株检测的高灵敏度。
