A single tube nested PCR for the detection of hepatitis C virus RNA

An assay was developed for the detection of hepatitis C virus RNA in serum which combined cDNA synthesis and a hot start nested polymerase chain reaction (PCR) in a single tube. This was made possible by separation of the reagents necessary for cDNA synthesis and PCR during cDNA synthesis with a high melting temperature wax interface and by the use of 'drop in-drop out' nested primers which enabled each primer set to be selectively extended within the same reaction tube. The increase in sensitivity following amplification with the internal primer pair was comparable to that achieved when nested reactions are carried out separately.