Allele-specific real-time PCR for the detection of minor HIV-1 variants

Abstract

Objective To develop and evaluate the allele-specific real-time PCR(ASPCR) assay for the detection of minor HIV-1 variants. Methods We developed and evaluated the ASPCR assay, using the K103N mutation site as a model system. We constructed plasmids as standards and designed specific and non-specific primers to discriminate the wild-type and mutant plasmids in the real-time PCR using SYBR green as fluorescence reporter. And then we evaluated the sensitivity, accuracy, reproducibility of ASPCR assay and detected the control samples. Results The specific primer can discriminate the wild-type and mutant plasmids including resistant mutation successfully. The sensitivity of ASPCR assay can achieve less than 0.01% and the accuracy of this method is down to 0.1%. The Intra-assay coefficient of variation is less than 0.7 and the Inter-assay coefficient of variation is less than 1.6. Conclusion ASPCR is a sensitive, accurate and rapid method to detect the minor HIV-1 variants which have resistant mutations and it can be used widely in HIV research. ASPCR also can provide earlier and more resistant information to the clinical therapy. Key words: HIV; ASPCR; Minor populations; Drug resistance