A convenient approach to the generation of multiple internal control DNA for a panel of real-time PCR assays.

Real-time polymerase chain reaction (PCR) assays allow convenient detection and quantitation of virus-derived nucleic acids in clinical specimens. When specimens are assayed for the presence of virus-derived nucleic acids against external standards, sample adequacy is not monitored. This can be achieved by using internal controls that are co-amplified with the virus-specific DNA in competitive PCR. Each of the various real-time PCR assays in a routine clinical laboratory requires its specific internal control. In order to complement a panel of virus-specific real-time PCR assays with internal controls, a convenient approach is described to generate the several internal controls within single DNA fragment. By applying composite primer technology, PCR primer sequences used in real-time PCR assays were added in 5' and 3' of a stretch of heterologous DNA during consecutive preparative PCRs. The heterologous DNA was used for internal control specific detection by e.g. FRET-hybridisation probes. The presented example of such a multiple internal control DNA contained five internal controls for five competitive LightCycler-PCR assays. All five PCR products derived from the multiple internal control DNA were detected with a single pair of specific FRET-hybridisation probes. The example described proved useful in real-time PCR assays specific for the detection of EBV-, CMV-, VZV- HSV-, and HBV-DNA on the LightCycler instrument. This methodology should enable laboratories to conveniently complement their panel of existing real-time PCR assays with a single multiple internal control DNA.