Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system

Abstract

We describe the application of a new fluorogenic probe-based PCR assay (TaqMan; Perkin Elmer Corp./Applied Biosystems, Foster City, Calif.) for the detection of hepatitis C virus RNA in serum and plasma. This assay allows for the direct direction of specific PCR products within minutes of completion of the PCR by monitoring the increase in fluorescence of a dye-labeled oligonucleotide probe. We evaluated this assay by comparing the results obtained by nested PCR with those obtained by TaqMan PCR. Test samples included two separate dilutions series of plasma samples from experimentally infected chimpanzees and a panel of 48 serum specimens from patients with community-acquired hepatitis C virus. The quantity of HCV RNA in each chimpanzee plasma sample was determined by using branched DNA (bDNA) signal amplification assay (Quantiplex HCV RNA assay; Chiron Corp., Emeryville, Calif.). Both PCR assays demonstrated similar levels of detection and could reliably detect 13 bDNA genome equivalents per sample. We found an overall concordance of 88% between results of two PCR assays with the community-acquired panel, which resolved to 100% when discrepant samples were retested by nested PCR. TaqMan compared favorably with nested PCR with key advantages of speed, increased throughput, and decreased opportunity for false-positive results because of elimination of second-round amplification.