To identify hepatitis C virus (HCV) infection, HCV genomic ribonucleic
acid (RNA) can be detected using the reverse transcription-nested pol
ymerase chain reaction (RT-nested PCR). HCV replication involves the p
roduction of a complementary, genomic-length, negative RNA strand via
semiconservative RNA synthesis, utilizing negative strand specific rev
erse transcription and subsequent DNA synthesis. It is important to ex
clude the presence of self-priming in negative strand specific RT-nest
ed PCR assay. In these experiments, HCV genomic RNA was subjected to r
everse transcription without addition of a primer, and the resultant c
omplementary deoxyribonucleic acid (cDNA) was produced. Digestion of t
emplate with RNase suggests that the template RNA is reverse transcrib
ed with an RNA primer, not a DNA primer. Therefore, caution must be em
ployed in interpreting studies of HCV replication using negative stran
d specific reverse transcription and PCR.