Genex For the Analysis of QPCR And DPCR Data in Regulated Environments

Genex For the Analysis of QPCR And DPCR Data in Regulated Environments

Abstract
Quantitative real-time PCR (qPCR) and digital PCR (dPCR) have been around for more than 30 years and are the preferred molecular methods for analysis of nucleic acids. Their use is conceptually simple and the actual analyses are quite straightforward to perform, which rapidly made the methods exceedingly popular. However, analyzing complex biological samples is much more challenging than performing only the qPCR and dPCR analytics, which led to high rate of erroneous studies and even misuse in the early days. The MIQE guidelines were published in 2008 and the dMIQE guidelines in 2020 to guide researchers and reviewers of academic publications what information about qPCR and dPCR shall be reported to make proper assessment of the experiments possible to readers. These publications dramatically improved the confidence level of publications that were compliant. qPCR and dPCR became also the preferred methods in molecular diagnostics and, indeed, dominated totally the testing of SARS-Cov-2 virus during the recent pandemic. This global testing, however, also revealed the complexity of calibrating and standardizing molecular analysis methods. To support these efforts the ISO standard “Requirements for evaluating the performance of quantification methods for nucleic acid target sequences — qPCR and dPCR” was published. Most recently qPCR and dPCR are also becoming the preferred methods for Pharmacokinetic, Pharmacodynamic and Biodistribution studies in clinical trials of Cell and Gene Therapeutics (CGT) and Advanced Therapies and Medicinal Products (ATMP). The calibration and validation of qPCR and dPCR assays require determining performance parameters such Limit of Detection (LoD), Limit of Quantification (LoQ), Dynamic range, Linearity, repeatability, reproducibility and sometimes also robustness, ruggedness and more. GenEx software has been around for more than 20 years and constantly developed to include new tests and analyses and to be compliant with requirements from regulators. Some methods, such as the determination of LoD and LoQ for qPCR assays, were even developed by our team. In this talk we present workflows to validate and characterize the performance of new assays and exemplify their use in SARS-Cov-2 diagnostics and in clinical trials.