Universal profiling of HIV-1 pol for genotypic study and resistance analysis across subtypes.

摘要

BACKGROUND: The increased use of anti-HIV-1 treatments in developing countries primarily infected by non-B subtypes necessitates development of novel tools to assess susceptibility and resistance. HIV-1 genomes are highly polymorphic and present challenges for the development of universal protocols capable of screening across subtypes. Currently available viral genotyping methods are useful for viral quantification, but are inadequate for sequence profiling or comprehensive mutation detection in the variable regions of HIV polymerase (pol). METHODS: A novel set of universal primers within pol, with consensus among a variety of HIV-1 subtypes, was developed. One-round amplification was performed by one-step reverse transcription PCR on 79 samples from HIV-1 subtypes. Using a second set of primers, the amplified fragment was sequenced and assembled to produce a profile database per sample. RESULTS: First-round amplification using universal primers generated a unique amplicon encompassing the major pol regions in all tested HIV-1 subtype samples. Sequence analysis of the amplified fragment not only confirmed the subtype of each HIV-1 isolate but also identified resistance mutations in the pol genes of HIV-1, including protease, reverse transcriptase, connection, RNase H, and integrase. Last, some of these primers were used to develop a viral load test using quantitative real time-PCR. CONCLUSIONS: A novel protocol was produced to effectively identify and simultaneously generate extensive sequence profiles of pol genes across HIV-1 subtypes. This protocol allows for expeditious and cost-effective mutation detection, genotypic evaluation and viral load determination in multiple HIV-1 subtypes.