The use of melting curves as a novel approach for validation of real-time PCR instruments

Validation of PCR thermal cycler performance is crucial in order to obtain reliable results. In this study, high resolution melting curve (HRM) analysis is presented as a novel validation method for real-time PCR instruments. By applying HRM analysis using a defined PCR amplicon and EvaGreen dye, information about the temperature accuracy 1 and thermal homogeneity of the heating block was obtained. This pilot study shows the potential of our technique for temperature validation of real-time quantitative PCR thermal cyclers. Our data correlated well with the temperature accuracy data obtained from the Mobile Temperature Acquisition System (MTAS; r(2) = 0.93), which conforms to the National Institute of Standards and Technology criteria, and our method was reproducible in independent runs (r(2) = 0.95). The advantages of this HRM-based method include: (i) temperature measurement under real world conditions in the reaction liquid in closed reaction tubes; (ii) temperature measurement of all wells; and (iii) applicability to all real-time PCR instruments capable of HRM analysis.