Detection of hepatitis C virus RNA by a combined reverse transcription PCR assay: comparison with nested...

Many of the current reverse transcription (RT)-PCR assays for the detection of hepatitis C virus (HCV) RNA are multistep processes which use multiple enzymes and buffers. The assays are also often suboptimal, requiring nested amplification to achieve the desired levels of sensitivity. As a result, these assays are cumbersome and prone to false-positive results. The susceptibility to contamination is further aggravated by the lack of carryover controls. We have previously reported the development of a combined RT-PCR assay for HCV RNA detection which is sensitive and simple to perform. We have since successfully integrated dUTP-uracil-N-glycosylase carryover prevention into the combined assay. Restriction of as much as 0.5 microliter of deoxyuridine-containing amplification products has been achieved. The performance of the improved combined assay was compared directly with conventional nested RT-PCR and antibody detection. The combined assay was found to have sensitivity similar to that of nested RT-PCR in detecting HCV RNA from HCV antibody-positive specimens. In an analysis of hepatitis B virus antibody-positive specimens, nested amplification had false-positive rates ranging from 8 to 31%, while no false-positive results were seen with the combined assay. In comparison with serological methods, the combined assay had specificity and sensitivity of 100 and 95%, respectively.