We describe a simple and sensitive approach for mutant virus screening that is able to detect drug-selected resistance mutations at frequencies as low as 0.3% in clinical samples, allowing for the identification of minority HIV-1 variants. The real-time PCR-based point mutation assays were robust with the 474 total subtype B virus specimens evaluated, supporting their use for clinical testing. Improved low-frequency mutation detection was provided by clinical testing cutoffs that were 10–67-fold more sensitive than conventional sequencing. These cutoffs were above the background reactivities observed with drug-naïve wildtype HIV collected in the pre-antiretroviral drug era and, thus, identify mutations occurring at frequencies above those found naturally in virus quasispecies. Although this paper focused on resistance mutation testing in subtype B viruses, we earlier demonstrated that real-time PCR assays can also be successfully developed for subtype C viruses which are globally the most prevalent 
. When possible, oligonucleotides are designed so that they might also be used with more than one subtype; however, screening for resistance in non-B subtypes requires that the oligonucleotides are properly validated for those subtypes.
Setting stringent assay cutoffs to avoid detecting natural polymorphisms resulted in primer designs that provided sensitivities of 96% to >99% and specificities of >99% with samples that included highly polymorphic sequences (). Although the real-time PCR assays were able to detect as little as 0.001%–0.2% cloned mutant sequences, increasing ΔCT cutoffs to expand the mutation detection range would make it difficult to differentiate drug-selected mutants from naturally-occurring variants. However, in antiretroviral studies of infected persons in which pre-drug exposure samples are available, a comparative method could be used to evaluate drug resistance rather than an absolute ΔCT cut-off. In these settings, a substantial decrease in ΔCT between the pre- and post-treatment samples for an individual could indicate the emergence of a mutation even if the ΔCT does not drop below the cut-off established for screening. Furthermore, in experimental settings where baseline genotypes are known, individual primers that best match the virus sequence may be used, instead of mixtures, to maximize assay sensitivity.
Evidence of improved resistance mutation detection was found in testing only a few samples which uncovered hidden mutations. However, to overcome the limitation of single mutation detection, we directly sequenced mutation-specific reactions as a simple way to rapidly assess mutation associations and demonstrated that the genotypic findings were similar to that obtained by cloning virus templates. Sequencing mutation-specific amplicons also identified additional low-frequency drug resistance mutations when they were linked to the targeted mutation, as was seen with the discovery of M184V in sample B. Therefore, previously hidden multi-drug resistance could easily be uncovered.
Sensitive testing can be streamlined by using a tailored and concise panel of mutation-specific tests that span the protease and RT regions, followed by sequencing the mutation-specific amplicons from positive tests to evaluate for linked mutations. This would allow for sensitive primary screening of resistance as well as the identification of other mutations present in the individual. The capacity to identify linked mutations could be important for understanding the persistence 
and clinical impact of mutant variants.
In conclusion, we present a panel of real-time PCR assays that provide a sensitive and user-friendly method for screening HIV-1 drug resistance mutations. The substantial oligonucleotide modifications that allowed for successful detection of mutations within diverse sequence backgrounds, combined with extensive validation and improved sensitivity, make these assays feasible for large-scale resistance testing. Furthermore, coupling mutation-specific sequencing to sensitive screening expands the capability of point-mutation testing and provides a powerful approach for studying the dynamics and clinical consequences of drug-resistant HIV-1. The simplicity of this methodology and the abundance of real-time PCR materials currently make sensitive PCR assays more practical for broader drug resistance testing than the more complex and expensive testing methods.