Sensitivity of multiplex real-time PCR reactions, using the LightCycler and the ABI PRISM 7700 Sequence Detection System, is dependent on the concentration of the DNA polymerase

Abstract

The introduction of multiplex PCR techniques to clinical laboratories has provided a means to streamline assays and to produce multiple results with minimal effort. While this methodology is very beneficial, care must be taken to ensure that reactions are properly optimized to allow for maximum sensitivity. This study was conducted to determine whether the sensitivity of multiplex-real-time PCR assays could be improved by increasing the concentration of DNA polymerase within a reaction. Multiplex reactions were designed to simultaneously detect the human HLA-DQ gene and a sequence from the UL83 region of the CMV genome. Two real-time PCR systems, one utilizing AmpliTaq Gold DNA polymerase and the ABI 7700 Sequence Detection System, and one utilizing FastStart Taq DNA polymerase and the Roche LightCycler were tested. The results indicated that increasing the AmpliTaq Gold concentration from 0·050 to 0·10U/μl and the FastStart Taq concentration from 0·1875 to 0·375U/μl increased detection sensitivity from 5000 to 50CMV copies per PCR reaction. In separate experiments, commercially prepared mastermixes were utilized for both real-time PCR platforms as per the manufacturer's suggestions or with the addition of supplemental DNA polymerase. In assays designed to detect 4CMV genome copies per reaction, the addition of 2·5U of AmpliTaq Gold to TaqMan Universal Mastermix increased the detection rate from 21 to 67%, and the addition of 5U of FastStart Taq to FastStart DNA Master Hybridization Probes mastermix increased the detection rate from 17 to 56%. These results indicate that increasing the DNA polymerase concentration in multiplex real-time PCR reactions may be a simple way to optimize assay sensitivity.