Design and work-up of a new molecular diagnostic assay based on real-time PCR

In routine molecular diagnostics, real-time PCR has made a major impact because of the faster time to the result; decreased hands-on time; and virtual elimination of the major issue in the early days of PCR, sample contamination from previously amplified DNA. The shorter time to the result for a real-time PCR assay means that a new diagnostic application may be developed relatively quickly. This chapter discusses the fluorescent chemistries typically used in molecular diagnostic applications and the additional features, such as internal controls, which are highly desirable in these assays. After an assay has been developed, the assay's performance must also be evaluated before the assay can be implemented in the testing laboratory.