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

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CRF01-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、CRF01-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CRF07-BC和CRF08-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43份(欧美B11份，泰国B32份)，C、CRF01-AE、A和D亚型样品分别为54份、17份、3份和2份。其中C亚型的样品，有52份属于CRF07-BC和CRF08-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46份(85．2％)和CRF01-AE13份(76．5％)。另外，检测CRF07-BC和CRF08-BC重组株的引物特异性地检测到43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100％。虽然有时会有非特异扩增带，但一般不影响结果判断。结论 我们建立了一套简单快速的H1V-1亚型鉴定方法，不需基因测序，即可检测我国主要流行株B、C、CRF01-AE、CRF07-BC和CRF08-BC。该方法具有高度特异性和敏感性，可以作为初筛方法在我国及其他国家HIV-1实验室推广使用。

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

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.