Checklist for optimization and validation of real-time PCR assays
Article first published online: 19 MAY 2009
© 2009 Wiley-Liss, Inc.
Journal of Clinical Laboratory Analysis
Volume 23, Issue 3, pages 145–151, 2009
How to Cite
Raymaekers, M., Smets, R., Maes, B. and Cartuyvels, R. (2009), Checklist for optimization and validation of real-time PCR assays. J. Clin. Lab. Anal., 23: 145–151. doi: 10.1002/jcla.20307
- Issue published online: 19 MAY 2009
- Article first published online: 19 MAY 2009
- Manuscript Accepted: 4 MAR 2009
- Manuscript Received: 16 JUL 2008
- real-time PCR;
- molecular diagnostics;
Real-time polymerase chain reaction (PCR) is a frequently used technique in molecular diagnostics. To date, practical guidelines for the complete process of optimization and validation of commercial and in-house developed molecular diagnostic methods are scare. Therefore, we propose a practical guiding principle for the optimization and validation of real-time PCR assays. Based on literature, existing guidelines, and personal experience, we created a checklist that can be used in different steps of the development and validation process of commercial and in-house developed real-time PCR assays. Furthermore, determination of target values and reproducibility of internal quality controls are included, which allows a statistical follow-up of the performance of the assay. Recently, we used this checklist for the development of various qualitative and quantitative assays for microbiological and hematological applications, for which accreditation according to ISO 15189:2007 was obtained. In our experience, the use of the proposed guidelines leads to a more efficient and standardized optimization and validation. Ultimately, this results in reliable and robust molecular diagnostics. The proposed checklist is independent of environment, equipment, and specific applications and can be used in other laboratories. A worldwide consensus on this kind of checklist should be aimed at. J. Clin. Lab. Anal. 23:145–151, 2009. © 2009 Wiley-Liss, Inc.