Detection of HIV-1 M184V and K65R drug resistance mutations by Allele specific real-time PCR in patients on Anti-Retroviral Therapy (ART)

Abstract

Objective: The aim of this study was to design and evaluate an allele-specific real-time PCR based assay used to detect HIV-1 M184V and K65R drug resistance mutations in ART treated patients. Nucleotide polymorphisms are a major challenge in the development of point mutation PCR-based assays for the detection of HIV-1 drug resistance mutations which could be a cheaper alternative than bulk sequencing. The use of phosphorothioate modified primers and DNA polymerases with proof reading ability is a novel application in the detection of HIV-1 sequence variants or mutations at a particular location. The fidelity for primer extension of the Pfu DNA polymerase used in this study was compared to that of the Taq DNA polymerase, an enzyme that lacks 3’-5’ exonuclease proof reading ability and is commonly used in PCR reactions. Method: The K65R forward primer and the M184V reverse primer were designed such that the 3’ penultimate nucleotide corresponded with their respective point mutations (which confer drug resistance). The M184V reverse primer and the K65R forward primer were designed to incorporate a phosphorothioate modification at the 3’end of the primer to eliminate mismatch amplifications. The phosphorothioation was characterised by replacing sulphur with an oxygen atom within the nucleotide phosphate backbone which creates a bond that is resistant to cleavage by the 3’-5’exonuclease activity of the Pfu (Pyrococcus furiosus) DNA polymerase. Primers were designed to anneal with a melting temperature greater than 60ºC to ‘cure’ for the anticipated diminished binding exhibited by many primers as a result of HIV nucleotide polymorphisms. The allele-specific assay was optimised, tested on Virology Quality Assurance (VQA) panels and run on samples from patients undergoing antiretroviral therapy. Patient samples were tested using both M184V and K65R mutant and wild type phosphorothioate modified primers and the detected mutations were confirmed by bulk sequencing. Results and Discussion: From a total of 101 patients on ART who were screened for the presence of HIV-1 M184V and K65R drug resistance mutations by real-time allele specific PCR, five tested positive for presence of the M184V drug resistance mutation. Four of these were confirmed to harbour the mutation by bulk sequencing while one was negative (wild type). Among the five samples, only one tested positive for the presence of both M184V and K65R drug resistance mutations as determined by both real-time PCR and bulk sequencing. Although the discordant result between bulk sequencing and real-time PCR for one of the samples could not be quantitatively verified, it could possibly have been a result of minor population variants that could easily pass undetected by the bulk sequencing. Conclusion: This study provides a proof of principle for the use of modified primers in the detection of HIV-1 drug resistance mutations. This application could be used to develop cheap, sensitive assays that could detect HIV drug resistance mutations in a single reaction. Also, the phosphorothioate primer design may be useful in overcoming nucleotide polymorphisms that are known to interfere with point mutation PCR based assays.