Discrimination of lamivudine resistant minor HIV-1 variants by selective real-time PCR

A selective real-time PCR (SPCR) method was developed and evaluated for discrimination of resistance mutations in minor human immunodeficiency virus type 1 (HIV-1) populations, using the M184 mutation site as a model system due to its high clinical importance and the low genetic barrier to its development. The method enabled detection of minor viral populations down to 0.1%, and the relative proportions of different quasispecies could be easily displayed using cycle threshold (Ct) values. An excellent concordance was found when the assay was compared with direct sequencing and cloning results. The impact of mismatch between virus and primer/probe sequences was evaluated, showing that 3′ end mutations in the selective downstream primers were very disruptive and that 5′ end polymorphisms in the probe area were directly fatal, while mutations in the middle or the 3′ end of the probe were less disruptive. These effects were compensated by introducing wobble bases to accommodate the mutations. This sensitive and reliable point-mutation assay, analyzing M184I/V and other important mutations, will be fruitful in gaining new scientific knowledge about the kinetics of resistance mutations in minor viral populations of HIV-1 infected patients at failure of antiretroviral therapy.