Specific detection of minus-strand hepatitis C virus RNA by reverse: Transcription polymerase chain reaction on PolyA+-purified RNA

A full-length complementary DNA (cDNA) clone of the hepatitis C virus (HCV) genome was used to prepare full-length plus- and minus-strand RNA. The minus-strand RNA, which contains a polyA tract complementary to the polyU tract found in the plus strand (genomic) RNA, but not the plus strand RNA, was captured with a commercial polyA tract isolation system. After elution, the minus strand was amplified by reverse-transcription polymerase chain reaction (RT-PCR). The combination of this procedure and RT-PCR using rTth resulted in an unprecedented level of discrimination of 10 logs. HCV minus-strand RNA isolation was unaffected by the addition of an excess of 10 of plus strands or by the addition of cellular RNA, and although the polyA isolation step removed 99.99% of plus strands, there was no loss of minus-strand signal. Minus-strand RNA was detected in RNA extracted from 4/4 liver samples and 4/8 peripheral blood mononuclear cells (PBMC) samples examined. Because the titer of plus-strand HCV RNA in any sample makes a significant contribution to false, random, and self-priming, removal of the plus strand in this manner results in the most accurate method yet devised to confirm the replication of HCV in a population of cells