Single step PCR with a sensitivity similar to nested PCR for the detection of hepatitis C virus RNA

Objective. Evaluation of the performance of different HCV PCR detection systems for HCV RNA. A nested PCR, considered the reference assay, was compared with two single-step methods (ss-PCR) : the first is based on the detection of PCR products by liquid hybridization with a 32 P end-labelled probe (isotopic ss-PCR), while the second assay is a colorimetric method (colorimetric ss-PCR) using microwell plate hybridization with a specific nucleic acid probe (Amplicor HCV PCR, Roche Diagnostics Systems). Methods. Sera from 56 patients with suspected hepatitis C infection based on reactive serology or altered liver parameters, and sera from 15 blood donors were tested for HCV RNA. After RNA extraction, the synthesized HCV cDNA was amplified in parallel using isotopic ss-PCR, colorimetric ss-PCR and nested PCR. The products were detected by autoradiography, color development and ethidium bromide fluorescence, respectively. Results. In order to assess the analytical sensitivity of ss-PCR versus that of nested PCR, experiments included serial dilutions of positive control samples. Results showed that both methods had an extinction signal at the 1 :512 dilution. A comparative analysis of 71 clinical sera samples was obtained using the three protocols and the results clearly documented 100% concordance. Conclusions. Single step PCR methods for HCV RNA have a sensitivity equal to that of nested PCR and appear more suitable for diagnostic applications. Ss-PCR is safer than nested PCR in terms of both specificity and contamination problems. In particular, the Roche Amplicor HCV PCR assay minimizes sample exposure and management problems.