EVALUATION OF PCR AND NESTED PCR FOR LABORATORY DIAGNOSIS OF HEPATITIS-C VIRUS-INFECTION

The detection of hepatitis C virus (HCV) RNA by nested polymerase chai n reaction (PCR) is believed to be the most reliable method to diagnos e HCV infections.(1,2) A pitfall of nested PCR is that it is prone to contamination. Single step reverse transcription-PCR (RT-PCR) was perf ormed, prospectively, on 80 sera from 59 patients with a set of primer s that amplified a 273 bp sequence unique to the 5' noncoding (NC) reg ion of the HCV genome. Nested PCR, was performed on all PCR negative s pecimens with a set of primers that amplified a 255 bp internal to the original primers. Single step RT-PCR was positive on 45 sera from 35 patients following gel electrophoresis and on two additional sera from two patients following Southern blot hybridization. Nested PCR was po sitive on two more sera following gel electrophoresis of the nested PC R products. These two patients were seropositive and subsequent serum from one patient was positive by single step PCR. Three additional ser a were positive following Southern blot analysis of the nested PCR pro ducts. Two patients were seropositive and had elevated serum alanine a minotransferase (ALT) levels. The third patient was seronegative with normal ALT level and was considered a false positive. The remaining se ronegative control specimens were PCR negative by both methods. The ma jority of PCR positive patients (82%) had elevated ALT levels, while t he majority of PCR negative seropositive patients had normal ALT level s. We conclude that single step PCR is a sensitive test for the labora tory diagnoses of the majority of the HCV infections. (C) 1996 Academi c Press Limited