Veterinary PCR Diagnostics

Design and Optimization of Real-Time PCR Assays

Author(s): Raymaekers Marijke

Pp: 18-32 (15)

DOI: 10.2174/978160805348311201010018

* (Excluding Mailing and Handling)


With increased use of real-time polymerase chain reaction technology in molecular diagnostics, consistent procedures for design, optimization and validation of molecular diagnostic methods are needed. This chapter describes a practical guiding principle that can be used in different steps of the design and validation of in-house developed real-time PCR assays. The use of the described guidelines leads to more efficient and standardized optimization and validation. Ultimately, this results in a reliable and robust molecular diagnostic assay. A statistical follow-up of the performance of the assay is included and can be achieved by determination of target values and reproducibility of internal quality controls. Since this guiding principle is independent of environment, equipment and specific applications, it can be used in any laboratory.

Keywords: Oligonucleotide design; in-house assay; target gene; optimization; validation; specificity; melting curve analysis; amplicon sequencing; linearity; annealing temperature; internal quality control.

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