A RAPID AND HIGHLY SPECIFIC TECHNIQUE TO DETECT HEPATITIS-C RNA IN FROZEN-SECTIONS OF LIVER

Aims-To develop a polymerase chain reaction (PCR) based technique that would permit the rapid and highly specific detection of hepatitis C v irus (HCV) RNA extracted from frozen liver tissue. Methods-Samples of liver tissue from 18 patients undergoing orthotopic transplantation we re studied. patients were HCV positive. Total RNA was extracted from b etween one and 10 sections, 10 mu m thick, from each tissue sample. HC V RNA was amplified by (1) conventional, multistep reverse transcripti on PCR (RT-PCR) and by (2) combined, single step RT-PCR using coupled oligonucleotide primers based on the sequence of the 5' untranslated r egion of the viral genome. Positive results were confirmed by dot blot analysis using a digoxigenin labelled oligoprobe (Air 89). Results-HC V RNA was detected in the nine HCV positive patients by both conventio nal and combined RT-PCR. HCV RNA was not detected in the HCV negative patients. As little as 500 ng total RNA was needed as the template to yield detectable amounts of amplified cDNA. The digoxigenin labelled o ligoprobe hybridised with HCV RNA positive specimens only. Conclusions -The combined, single step RT-PCR is a rapid and sensitive technique f or detecting HCV RNA in frozen liver tissue.