Abstract Highly Active Anti-Retroviral Therapy (HAART) has effectively inhibited the prevalence of HIV-1 and reduced the death rate caused by AIDS. In recent years, the emergence of resistance-conferring RT gene mutations in HIV-1 strains has become the major reason for HAART failure. The detection of drug resistance is important for the HAART regimen choice and novel drug development. In this work, a novel assay for the detection of HIV-1 RT drug resistance mutations was developed. HIV-1 drug resistance and wild strains in B subtypes were investigated using Two-Step Mutagenically-Separated PCR (MS-PCR), and point mutations including M41L, K70R, K103N, Y181C, T215F were detected. We designed a longer mutant type primer, using microplates hybridization and ELISA technique to detect several point mutations within a mixed mutant-wild type population. The results indicate that the Two-Step MS-PCR is as sensitive and specific as that in the traditional MS-PCR and MS-PCR combined with ELISA can give a good P/N quotient with better sensitivity, low cost, relatively less time consumption and high-throughput screening. We suggest that it will be used in clinic usage for the detection of HIV-1 drug resistance mutations as well as other point mutations.
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