Development of a one-tube multiplex reverse transcriptase-polymerase chainreaction assay for the simultaneous amplification of HIV type 1 group M gag and env heteroduplex mobility assay fragments

The emergence of intersubtype recombinant HIV-1 isolates has made it impera tive to analyze different regions of HIV-1 genomes. For this purpose a one- tube multiplex RT-PCR, coamplifying first-round amplicons that allow amplif ication of gag and env heteroduplex mobility assay (HMA) fragments from dif ferent 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 p ositive for both gag and env, positive amplification of the gag fragment wa s 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 econom ical.