583 New Real-time RT-PCR Assay Quantifies K103N NNRTI-resistant Variant at a Frequency as Low as 0.01% S. Palmer1, V. Boltz*1, F. Maldarelli1, E. Halvas2, J. Mellors2, J. Coffin1 1HIV Drug Resistance Prgm, NCI, NIH, Frederick, MD and 2Univ of Pittsburgh, PA
Background: Minor populations of drug-resistant HIV-1 variants present prior to therapy have been hypothesized to be a cause of treatment failure. It has not been possible to test this hypothesis because of the limited sensitivity of assays for rare variants. Standard population-based DNA sequencing methods cannot reliably detect HIV-1 variants that constitute <10% of the virus population. More sensitive and reproducible assays that detect minor HIV-1 populations are needed. We have therefore developed a high-throughput real-time RT-PCR assay to quantify minor populations of HIV-1. We have focused our initial efforts on detecting the K103N NNRTI-resistance mutation.
Methods: HIV-1 RNA is extracted from 0.250 to 0.500 ml of sample, and converted to cDNA using random primers. The target sequence region is amplified and quantified then diluted to approximately 107copies/reaction, and used as template for a second round of PCR, which is performed using primers that discriminate between the K or N allele. Second round PCR product is detected using SYBR green fluorescence. To confirm amplification specificity, PCR products are subjected to thermal denaturation analysis.
Results: Plasmid-derived wild type and mutant viruses were mixed in HIV-negative human plasma to a concentration of 0.5 million RNA copies/ml. The percentage of 103N mutant in the mixtures ranged from 100% to 0%. Blinded testing of the virus mixtures showed that the assay accurately quantified the frequency of the 103N variant from 100% to 0.01% (to within a factor of 2 of the predicted value) with an assay background of 0.006%. This background is not different from the single-step mutation rate and may reflect mutations arising during reverse transcription or first round PCR amplification. Analysis of assay reproducibility showed that the mean coefficient of variation averaged 31% across the range of mutant frequencies.
Conclusions: We have developed a real time PCR assay that permits the detection and quantification of the 103N variant at a frequency as low as 0.01% in cloned virus mixtures. This assay will help provide new insights into the prevalence of K103N minority species in HIV patients and the possible source of persistent viral replication or resistance in patients on antiretroviral therapy containing NNRTI inhibitors.
