ABSTRACT
A multiplex DNA PCR assay was developed for the simultaneous first-round amplification of HIV-1 gag and env fragments for the heteroduplex mobility assay (HMA). This assay was compared with the conventional amplification assay, using DNA extracted from PBMC samples from 30 HIV-1-seropositive individuals from The Gambia, who were enrolled between 1992 and 1997. From 27 of 30 (90%) samples both gag and env HMA fragments were amplified simultaneously. In one sample only the gag HMA fragment could be amplified by multiplex DNA PCR, and in two samples amplification was negative for both gag and env HMA in multiplex as well as the mono-DNA PCR. Of the 28 Gambian isolates subtyped by gag/env HMA or by sequencing and phylogenetic analysis, the majority (19 of 28; 68%) were intersubtype recombinant. Fifteen of 28 (53%) samples were circulating recombinant form (CRF) CRF02.AG variants. Two isolates clustering with the previously documented Gambian isolate GM4 (previously described as an env GC recombinant) are classified as gag A/env J recombinants.
Subject(s)
Genes, env , Genes, gag , Genetic Variation , HIV-1/classification , Polymerase Chain Reaction/methods , DNA, Viral/analysis , Gambia/epidemiology , HIV Infections/epidemiology , HIV Infections/virology , HIV-1/genetics , Heteroduplex Analysis , Humans , Molecular Sequence Data , PhylogenyABSTRACT
The emergence of intersubtype recombinant HIV-1 isolates has made it imperative to analyze different regions of HIV-1 genomes. For this purpose a one-tube multiplex RT-PCR, coamplifying first-round amplicons that allow amplification of gag and env heteroduplex mobility assay (HMA) fragments from different HIV-1 group M isolates, was developed, starting with plasma samples. The multiplex RT-PCR assay is sensitive: 115 of 136 (84.5%) samples were positive for both gag and env, positive amplification of the gag fragment was observed in 130 of 136 (95.6%) samples, while for the env fragment 119 of 136 (87.5%) tested positive. The multiplex RT-PCR in combination with gag and env HMA makes large-scale HIV-1 subtyping fast, simple, and more economical.
