A well-designed and -optimized polymerase chain reaction (PCR)-based assay has the potential to be a highly sensitive and highly specific nucleic acid target detection method, relative to other diagnostic methods. However, even good PCR assays have limitations as there are many factors which have to be controlled to ensure the success of a PCR run. As such, problems encountered in PCR runs require careful analysis and stepwise investigation, considering the said factors. This guidance document outlines the factors influencing the outcome of Taqman-based Real-Time PCR runs, the common problems/challenges encountered, practical troubleshooting procedures, and provides recommendations on how to address these issues.
Error recognition and correction are important and these should be integrated in the laboratory’s Quality Management System (LQMS), spanning the pre-analytic, analytic and post-analytic phases of testing, to ensure that the final output – laboratory test results – are as accurate and reliable as possible.