Real time PCR assays for the detection of viral pathogens - are they the gold standard?

Over the past decade, PCR testing for infectious agents in the clinical laboratory has begun to replace other methods, such as culture. PCR is often now referred to as the 'gold standard' in this field. Further evolution of this technology has seen the introduction of real time PCR, replacing traditional gel detection methods for amplified products. At ARUP we have developed a number of real time PCR assays on the LightCycler (Roche) and the HT7900 (Applied Biosystems), using both one and two probe systems. During the development of these assays, we have encountered a number of unanticipated obstacles associated with real time PCR testing. Here we present data illustrating a few of the issues to keep in mind when setting up a real time PCR assay.