Two-round rapid-cycle RT-PCR in single closed capillaries increases the sensitivity of HCV RNA detection and avoids amplicon carry-over.

Abstract

For the detection of hepatitis C virus (HCV) specific nucleic acids the polymerase chain reaction (PCR) is widely used. Rapid-cycle PCR is performed in glass capillaries with the LightCycler instrument and allows PCR including product analysis to be performed within a closed system in about 1 h. Thus, rapid-cycle PCR appears especially suitable for routine diagnostic applications. However, the volume of the PCR vessel is restricted to about 20 microl, which may limit the sensitivity of the PCR. To increase its sensitivity two-round or nested primer PCR protocols have been developed. In rapid-cycle PCR first-round PCR products are usually collected from the capillaries by centrifugation, a procedure prone to cross-contamination.