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 min us-strand RNA, which contains a polyA(+) tract complementary to the polyU t ract found in the plus strand (genomic) RNA, but not the plus strand RNA, w as captured with a commercial polyA(+)-tract isolation system. After elutio n, 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(10). HCV m inus-strand RNA isolation was unaffected by the addition of an excess of 10 (4) of plus strands or by the addition of cellular RNA, and although the po lyA+ isolation step removed 99.99% of plus strands, there was no loss of mi nus-strand signal. Minus-strand RNA was detected in RNA extracted from 4/4 liver samples and 4/8 peripheral blood mononuclear cells (PBMC) samples exa mined. Because the titer of plus-strand HCV RNA in any sample makes a signi ficant contribution to false, random, and self-priming, removal of the plus strand in this manner results in the most accurate method yet devised to c onfirm the replication of HCV in a population of cells.