ABSTRACT
Since the invention of polymerase chain reaction (PCR) in 1983,1 much research has been done on the impact of the different variables within the pre-PCR and PCR process on the nal PCR result. This research has mainly focused on the impact of (1) sampling methods,2 (2) RNA and DNA isolation and purication procedures,3,4 (3) quality and purity of RNA, cDNA, and DNA, and ways to determine this,5,6 (4) primer and probe design strategies, (5) primer and probe quality and purity,7 (6) RT-and PCR-priming strategies,6 (7) detection chemistries,6,8-10 (8) types of RT and PCR enzymes,11,12 (9) PCR buffer compositions,12 and (10) PCR enhancers and inhibitors.13 Yet, not much research has been done on the impact of thermocyclers on the PCR result. Thermocyclers are considered to be constants rather than variables, although most researchers are familiar with the effect that particular PCRs function well on certain thermocyclers and fail, or generate different results, on others.14-16
With the more recent use of PCR in diagnostics, the call for quality control (QC) is increasing in accredited-and quality-aware laboratories. An increasing number of laboratories either elect, or are required, to obtain an ISO 1702517 or ISO 1518918 accreditation to guarantee the quality of the results generated. At the same time, the research community’s call for biologically meaningful
9.1 Introduction .......................................................................................................................... 125 9.2 Terms and Denitions ........................................................................................................... 126 9.3 Thermocycler Technical Design ........................................................................................... 127
9.3.1 Various Technologies ................................................................................................ 127 9.3.2 Speed versus Uniformity .......................................................................................... 127
9.4 Thermocycler Variability and Practical Consequences ........................................................ 128 9.4.1 Inter-and Intra-Thermocycler Variability ................................................................ 128 9.4.2 Consequences of Thermocycler Variability ............................................................. 129
9.5 Effect of Thermocycler Variability on a PCR or qPCR ....................................................... 130 9.6 Thermocycler Calibration Methods ...................................................................................... 131 9.7 Evaluating Calibration Results ............................................................................................. 132 9.8 Modifying Thermocycler Performance ................................................................................ 136 9.9 Assay Validation ................................................................................................................... 137
9.9.1 Introduction .............................................................................................................. 137 9.9.2 Hot-Cold Spot Validation Method ........................................................................... 138 9.9.3 Thermal Boundary Validation Method .................................................................... 138 9.9.4 Approximated Thermal Boundary Validation Method ............................................ 140 9.9.5 CE-IVD Kit Validation, Verication, and Revalidation ........................................... 140
9.10 Conclusions ........................................................................................................................... 141 References ...................................................................................................................................... 142
conclusions is increasing in parallel. In 2009, a group of leading qPCR scientists published the MIQE guidelines,19 which assist qPCR users in designing a robust qPCR experiment that leads to trustworthy and biologically meaningful results that can be reproduced in any other laboratory. As a result of this call for QC, the number of thermocycler calibrations and assay-thermocycler validations has strongly increased. Through these calibrations, it has become clear that there is a substantial variation not only between different brands of thermocyclers, but also between models of the same brand, between individual serial numbers of one model, and also within one thermocycler. This variation can have a substantial impact on the outcome of PCRs or qPCRs. The effects move on a sliding scale from slightly less-efcient PCRs to complete failure.
