DETECTION OF HEPATITIS-C VIRAL SEQUENCES IN SERUM BY NESTED POLYMERASE CHAIN-REACTION (PCR) AND A COMMERCIAL SINGLE-ROUND PCR ASSAY

Background: Demonstration of the hepatitis C virus (HCV) genome is usu ally done with combined reverse transcription and polymerase chain rea ction (RT-PCR) employing nested primer sets. Recently, a commercial PC R assay (Amplicor PCR assay), based on a simplified sample preparation procedure, a single, combined reverse transcription and polymerase ch ain reaction (RT-PCR), and a microwell plate capture and detection, ha s been developed. Objective: The aim of the present study was to compa re the new Amplicor assay with an 'in-house' PCR. Additional testing i ncluded a third-generation enzyme immunoassay for anti-HCV antibodies, the Wellcozyme HCV Western Blot, which is equivalent to a third-gener ation recombinant immunoblot assay. Furthermore, HCV genotypes were cl assified. Study design: Sera from a total of 127 patients were studied . After screening with a third-generation enzyme immunoassay (EIA), th e Wellcozyme HCV Western Blot, was performed as well as the convention al RT-PCR and the Amplicor PCR. Specimens, which were found positive b y testing with the Amplicor kit, were subjected to storage at room tem perature for 96 h. Results: A total of 52 patients were found to be po sitive for anti-HCV by the third-generation ELA. With the Amplicor ass ay, the HCV genome was detected in 38 patients. In comparison with the 'in-house' assay, two discrepant results were found. Resolution of di screpant samples increased the total number of true positives to 39. A good correlation was found between a positive anti-HCV test result an d the presence of HCV-RNA by RT-PCR. No significant reduction in the a mount of amplification product was observed by retesting of suboptimal ly stored samples with the Amplicor assay. Conclusion: Because of the rapidity and the improved ease of handling, the Amplicor assay was fou nd to be a good contribution for detection of HCV in serum.