Development of selective real-time PCR (SPCR) asays for the detection of K103N resistance mutation in minor HIV-1 populations

[摘要] ENGLISH ABSTRACT: Background: The conventional sequence analysis is the most common method used for the detection of drug-resistant mutants. Due to its sensitivity limitations, it is unable to detect these mutants when comprising less than 20% (minor populations) of the total virus population in a sample. However, real-time PCR-based assays offer a rapid, sensitive, specific and easy detection and quantification of such mutants. The HIV-1 variants harbouring the K103N mutation are associated with resistance to nevirapine (NVP) and efavirenz (EFV). The persisting drug-resistant mutants decay slowly to low levels, and therefore they are called minor drug-resistant mutants. Consequently, they affect subsequent treatment with the drugs of the relevant class.Objectives: The objective of this study was to design two TaqMan real-time PCR-based assays called selective-polymerase chain reaction (SPCR), namely the total viral copy SPCR assay and the K103N-SPCR assay. The former detects HIV-1 of subtype C reverse transcriptase sequences, whereas the latter detects K103N drug-resistant variants in these sequences.Design and Methods: In developing the SPCR assays, sets of appropriate primers and probes for the HIV-1 subtype C reverse transcriptase (RT) were developed to use in the K103N-specific reaction and the total copy reaction. Twelve DNA plasmid standards with sequence diversity were constructed for the assay from two HIV-1subtype C samples known to harbour the K103N mutation (AAC or AAT) in our Department‟s Resistance Databank. Their RT regions were amplified, cloned and verified with sequencing. Site-directed mutagenesis was used to induce mutations at 103 amino acid position in some of these clones to generate more standards with either one of the three codons (AAA, AAC and AAT). The two assays were optimized and validated, and a standard curve was generated for each assay using 10-fold serial dilution (5x107-5x100 DNA copy/μL) of a K103N-mutant plasmid standard. The optimized and validated SPCR assays were used to screen 40 nested PCR products of previously genotyped patient samples for minor K103N variants.Results: Two sensitive and reproducible selective real-time PCR (SPCR) assays, with cut-offs of 8.23 and 10.33 and a detection limit of 0.01% for the K103N resistance variants, were successfully developed. The assays detected a prevalence of 25.64-46.15% for the K103N resistance mutation in 39 patient samples. The genotyping (population sequencing) missed 40-53.85% of these variants.Conclusion: In conclusion, sensitive and reliable selective real-time PCR assays to detect and quantify minor K103N variants of HIV-1 in nested PCR products were successfully developed. The assay had a lower detection limit of 0.01%.

[发布机构] Stellenbosch University