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(10). HCV minus-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 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.
Clinical Medical Virology Research Center, University of Queensland, St. Lucia, Australia.
S T
ST
Takyar
D s
Ds
Li
Y h
Yh
Wang
R
R
Trowbridge
E J
EJ
Gowans
D006526:Hepatitis C / Q000209:etiology; D006801:Humans; D011061:Poly A / Q000032:analysis* / Q000302:isolation & purification; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D012367:RNA, Viral; D011061:Poly A