Optimization of Human Cytomegalovirus LightCycler Real-Time PCR

Abstract

Background: Real-time PCR has been widely considered as a powerful tool for the evaluation of Human Cytomegalovirus (CMV) DNA kinetics. Successful PCR relies on optimization, which is an extremely demanding procedure. Nevertheless, certain values could be optimal for most primers in use. Methodology: Seventeen CMV primer sets recommended in the literature were selected for optimization in terms of MgCl₂ and primers concentrations as well as annealing temperature using the LightCycler instrument and SYBR Green I detection format. Optimal values were considered as those showing the lowest crossing point (Cp), the highest fluorescence intensity, the steepest sigmoid curve slope, and the absence of non-specific PCR products. Results: Optimal values for most studied primers were found to be 3 mM for MgCl₂ concentration, 0.5 μM and 0.6 μM for primers concentration, and 55ºC for annealing temperature. Conclusion: Adopting the resulting values for CMV-specific primers generally used in single-target real-time PCR assays with the same thermal cycler may guarantee their efficient performance minimizing cost and time needed for optimization.