FD V03-112 recommends that a thermocycler undergo a two-part verification: a metrological verification and a biological verification.
We propose a metrological temperature verification that covers all critical phases of a PCR cycle — denaturation, hybridization, and elongation — by measuring the temperature within a liquid placed in a microplate or microtubes. This approach allows for characterizing the accuracy of the effective temperature observed in the reaction medium, taking into account influential parameters such as:
• the temperature of the heated lid;
• the thermal coupling between the container and the wells of the heating block;
• the thermal inertia effect resulting from the combination of the support and the liquid.
This methodology provides a rigorous evaluation of the actual thermal profile encountered during cycles, which is essential for ensuring the reliability of amplification results and performance compliance. It is applicable to all non-rotary heating block thermocyclers, whether or not they integrate a temperature gradient functionality.
