DETECTION OF HEPATITIS-C VIRUS-RNA BY A COMBINED REVERSE TRANSCRIPTION PCR ASSAY - COMPARISON WITH NESTED AMPLIFICATION AND ANTIBODY TESTING

Many of the current reverse transcription (RT)-PCR assays for the dete ction of hepatitis C virus (HCV) RNA are multistep processes which use multiple enzymes and buffers. The assays are also often suboptimal, r equiring nested amplification to achieve the desired levels of sensiti vity. As a result, these assays are cumbersome and prone to false-posi tive results. The susceptibility to contamination is further aggravate d by the lack of carryover controls. We have previously reported the d evelopment of a combined RT-PCR assay for HCV RNA detection which is s ensitive 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 mu l of deoxyuridine-containing amplif ication products has been achieved. The performance of the improved co mbined 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 antibod y-positive specimens. In an analysis of hepatitis B virus antibody-pos itive specimens, nested amplification had false-positive rates ranging from 8 to 31%, while no false-positive results were seen with the com bined assay. In comparison with serological methods, the combined assa y had specificity and sensitivity of 100 and 95%, respectively.