Optimization of allele-specific PCR using patient-specific HIV consensus sequences for primer design

Abstract

Allele-specific PCR based on subtype consensus sequences is a powerful technique for detecting low frequency drug resistant mutants in HIV-1 infected patients. However, this approach can be limited by genetic variation in the region complementary to the primers, leading to variability in allele detection. The goals of this study were to quantify this effect and then to improve assay performance.

Figure 1

Comparison of allele-specific PCR efficiency of patient samples between primers based on HIV-1 subtype C consensus sequences and primers based on patient-specific HIV-1 consensus sequences at codon 103. The total amplification efficiency of patient samples 1–4 (Table 1) was calculated as the sum of the efficiencies of detection of the wild-type and mutant alleles. Shown are results using the uncorrected consensus primers (grey) and the corrected primers (black) with error bars representing standard deviations of triplicate observations for samples taken immediately before single-dose nevirapine (BL) and 2 and/or 6 weeks following treatment. Patient 1 has 2 data sets; the 1^st are samples amplified with primers that match the HIV-1 sequence infecting patient 1, the 2^nd are samples amplified with primers only corrected to match the sequence at codon 104 in patient 1.