Specific detection of minus-strand hepatitis C virus RNA by reverse-transcription polymerase chain reaction on polyA+-purified RNA

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Abstract
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 logs10. HCV minus-strand RNA isolation was unaffected by the addition of an excess of 104 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.