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.