使用多重巢式PCR对我国HIV-1主要流行株亚型鉴定方法的建立

Development of a subtype screening assay for human immunodeficiency virus type 1 by nested multiplex PCR

目的 建立一套新的亚型鉴定方法 ,仅仅使用巢式PCR ,一次扩增 ,即可对我国HIV 1主要流行株B、C和CRF0 1 AE进行亚型鉴定。方法 从HIV阳性样本中提取核酸 ,使用能覆盖HIV 1型M组gag区的引物进行第一轮扩增 ,第二轮扩增则使用分别检测B、C、CRF0 1 AE亚型的三套特异性引物进行扩增 ,三套引物放在同一个反应管中。反应产物经琼脂糖电泳后观察 ,不同亚型的位置不同 ,以此来判断亚型。另外设计一套引物 ,专门检测我国重组株CRF0 7 BC和CRF0 8 BC。所有样品均经过基因测序、系统进化树分析 ,以进行结果验证。结果 在检测的 119份样品中 ,经基因测序和系统进化树分析证实B亚型样品 4 3份 (欧美B 11份 ,泰国B 32份 ) ,C、CRF0 1 AE、A和D亚型样品分别为 5 4份、17份、3份和 2份。其中C亚型的样品 ,有 5 2份属于CRF0 7 BC和CRF0 8 BC。而经过上述多重巢式PCR方法检测到的B亚型样品为 35份 (81 4 % ) ,C亚型 4 6份 (85 2 % )和CRF0 1 AE 13份(76 5 % )。另外 ,检测CRF0 7 BC和CRF0 8 BC重组株的引物特异性地检测到 4 3份 (82 7% )样品。上述结果与基因分析结果吻合 ,各个亚型之间无交叉 ,一种亚型的特异性引物只对该亚型有反应 ,而对其他亚型无反应 ,特异性达到 10 0 %。虽然有时会有非特异扩增带 。

Objective The current available assays for HIV subtyping,such as sequence-based phylogenetic analysis or heteroduplex mobility assay (HMA),are labor-intensive and time-consuming. The authors have just developed a simple and rapid subtype-screening assay for subtypes B,C,and CRF01-AE using a single nested multiplex PCR. Methods Proviral DNA from HIV-positive samples was extracted and subjected to first round PCR with universal primers for gag region that can detect HIV-1 M group isolates. In the second round PCR,three pairs of subtype-specific primers,respectively detecting subtype B,C and CRF01-AE,were added into one tube. The PCR products of different subtypes could be distinguished in agarose-gel electrophoresis. Another pair of primers exclusively detecting the prevalent recombinant B/C strains CRF07-BC and CRF08-BC were designed and used. Additionally,all of these samples were sequenced and analyzed phylogenetically. Results Phylogenetic analysis showed that out of 119 samples, there were 43 subtype B samples (Euro-American B 11,Thailand B 32),54 subtype C,17 CRF01-AE,3 subtype A and 2 subtype D samples. The subtype B,C,and CRF01-AE specific primer sets detected 35 (81.4%),46 (85.2%),and 13(76.5%) samples with accuracy and specificity. Non-specific bands occasionally appeared but did not interfere with interpretation of the results. The primer pairs for CRF07-BC and CRF08-BC amplified target sequences were confirmed by sequencing and phylogenetic analysis. The specificity of all these subtype-specific primers was found to be 100%. Conclusion A simple and rapid assay was developed for screening subtypes B,C,CRF01-AE,CRF07-BC and CRF08-BC in China. This assay may have potential application in HIV laboratories in China and worldwide.