Rapid identification for gag region of circulating recombinant form of humanimmunodeficiency virus type 1

ObjectiveTo develope a simple and rapid subtype-screening assay for the gagregion of the circulating recombinant form(CRF)of human immunodeficiency virus type 1(HIV-1)in Guangxi.MethodsProviral DNA from HIV-positive samples were extracted and subjected to the first round PCR with universal primers for the gag region that can detect HIV-1M group isolates.In the second round PCR,two pairs of subtype-specific primers that were designed to detect subtype C and CRF01-AE respectively were added into one tube.The PCR products of different subtypes could be distinguished in agarose-gel electrophoresis.Another pair of subtype-specific primers exclusively detecting the the prevalent recombinantstrains CRF07-BC and CRF08-BC was designed and used.Additionally,all of these samples were sequenced and analyzed phylogenetically.ResultsDNA sequencing and phylogenetic analysis of the gag region of the 54 samples showed that 4 samples(7.41%)were infected with CRF08-BC,46(85.18%)with CRF01-AE and 4(7.41%)remained unclassifiable.Detection of the subtype-specific primer sets revealed that 4 were B'/C(100%),and 45 were CRF01-AE (97.83%),with an adequate sensitivity(98%)and a high specificity(100%).Non-specific bands occasionally appeared but did not interfere with interpretation of the results.The phylogenetic analysis was consistent with subtype-specific primer sets and the consistent rate was 98.15%.The average reproducibility was 100% for B'/C samples and 98.3% for CRF01AE samples.ConclusionA simple,rapid and low cost assay is developed for subtype-screening of CRFO1-AE in Guangxi.For the B'/C strains in Guangxi,it needs to be verified further by increasing samples.