Abstract
The majority of HIV-infected patients attending St Mary's Hospital are of UK origin, and are infected with viruses which align with subtype B sequences; however, many are of African origin and infected with viruses of subtypes A, C or D. Increasingly, these patients are monitored for the effects of complex therapeutic regimens by sequencing the pol gene of HIV-1 from plasma to detect resistance-associated mutations. These tests use PCR amplification of HIV-1 RNA, or proviral DNA. We wished to determine whether sequencing primers used in HIV Genotyping System Version 2.0 (PE Biosystems), and seven published diagnostic and research-based nested DNA-PCR protocols used in the laboratory, covering 5' LTR, gag, pol, and env open-reading frames are able to amplify diverse genetic subtypes. Peripheral blood mononuclear cells were acutely infected with a panel of HIV-1 isolates representing env subtypes A to G (pol subtypes A, B, C, D and A'). RNA and DNA was extracted for RT-PCR and DNA-PCR and amplified according to protocols. The HIV Genotyping System Version 2.0 generates a 1.57kb amplicon of pol, which is then sequenced using three pairs of forward and reverse primers. This assay was able to amplify all subtypes tested. For the seven laboratory protocols, we accurately predicted the success or failure of each primer pair by comparison of primer binding to consensus sequence templates for each subtype (Los Alamos Database). These data emphasise the importance of ensuring primer sequences used in routine tests are regularly checked against new and divergent subtypes occurring in the local patient population. This subtype panel is a valuable resource, itself needing to be updated regularly to include new divergent strains.