Kenneth E. Pierce, Lawrence J. Wangh

Detection and identification of highly variable viral sequences is important for tracking infectious outbreaks and determining treatment regimens using targeted drug therapy. This report describes a single tube assay that is able to distinguish extensive sequence variation in hepatitis C virus (HCV) by using mismatch tolerant probes to analyze single-stranded amplicons generated with reverse transcription linear-after-the-exponential PCR (RT-LATE-PCR). Detection and identification of sequences from the 5' non-coding region (NCR) of 31 different HCV strains was first evaluated via hybridization of two fluorescently labeled, mismatch-tolerant probes to synthetic DNA strands. The resulting data were used to calculate the ratio of fluorescent signals for the two probes over a wide temperature range as well as the melting temperature (Tm) of each probe with the targets. Although the Tm measurements alone distinguished only 5 sequences from the others, fluorescent signal ratio analysis provided a unique set of values for 27 of the 31 strains. RT-LATE-PCR was then used to amplify Armored RNA (AR) containing the 5' NCR of five different strains of HCV. Melting analysis of the resulting single-stranded DNA with the two probes distinguished all five AR sequences. This assay can be expanded to include additional gene segments, and it points the way to construction of highly informative single-tube assays for HCV and other RNA viruses.