Allele-specific real-time PCR testing for minor HIV-1 drug resistance mutations: assay preparation and application to reveal dynamic of mutations in vivo.

Background It is very important for the clinical management to test for minor HIV 1 resistance mutations accurately and sensitively The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20%-30% The aim of this study was to evaluate allele-specific real time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215 Methods We developed the allele-specific PCR assay using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215FN as a model system The standards were constructed by cloning the wild-type and mutant DNA fragments into the T vector We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter And then we evaluated the ASPCR assay and tested 140 clinical samples using this method Results The sensitivities of ASPCR assay were 004% for K103N, 0 30% for M184I, 0 40% for M184V, 0 03% for T215F and 0 02% for T215Y The intra-assay and inter assay coefficients of variation were less than 0 42 One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained Conclusions Drug resistance emerged half a year after the start of antiretroviral therapy The mutation of T215Y emerged 1 to 1 5 years after starting treatment and then increased rapidly The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug resistance information for HIV research and AIDS antiretroviral therapy