How to validate cyclers?

Classical temperature sensors, like the thermometer and the thermocouple, do not meet modern requirements for accurate and reproducible temperature measurements on PCR instruments. By using thermistors CYCLERtest developed a dynamic and multichannel measurement system that is very well suited to monitor the performance of PCR instruments.

The MTAS system consists of 15 to 48 sensors, which, when placed in the sample position, measure the behaviour of a thermal cycler, simultaneously and with a frequency of 2 Hz per sensor. The signal is statistically and mathematically processed in the hardware and software of the system, to come to the most justifiable and precise result. The used set of sensors are matched very precisely to conform to the ITS-90 scale of the NIST (National Institute of Standards in Technology, Washington, USA). The performance of the system has been approved by international temperature accreditation bodies (ISO 17025 standard), and is defined to have an overall accuracy of better than 0.1°C. One of the strengths of the system is, without any doubt, that the performance test can be performed on location. The results are presented in extensive reports, and compared to field-specifications. The report includes a “Certificate of Thermal Performance”.

The two most important measured parameters are accuracy and non-uniformity of a PCR thermal cycler block. The accuracy shows the deviation of block temperature from set temperature, while the uniformity is a measure for the spread of temperature between wells of the same block. Both parameters depend on the temperature level at which they are determined; the accuracy of a thermal cycler normally decreases at elevated temperatures. In case the accuracy of a thermal cycler is known to be –1.0°C (this implies that the targeted temperature is not reached), one can take this into account while developing and optimising a protocol. For the parameter of non-uniformity this is much more difficult. However, the main advantage of a dynamic and multichannel measurement is that it is possible to check which wells deviate to much, and avoid the use of these hot or cold wells.

The speed (or ramp rate) of a PCR thermal cycler is an indication of the steepness of the slopes in between the temperature holds. Differences in ramp rate can influence the course of the reaction. Some cyclers are fast, but less controllable. The material of which the block is manufactured plays an important role. Directly related to speed, is the overshoot and undershoot parameter. This is the amount of °C that the target temperature is exceeded while trying to stabilise at a temperature hold. The only way to visualise this is by means of a fast, dynamic measurement.

While the MTAS measurement system is intended to certify the performance of a thermal cycler (e.g. yearly), DRIFTCON provides the opportunity to monitor the described parameters in time. The thresholds of a test can be determined empirically, and set as limits. So whenever these values are exceeded, it is not advised to perform the PCR test under these conditions.