b'Chapter 2 How should we assess PCR accuracy?An interview with Robin Patel, MDPCR may have entered the general lexicon during the pandemic,PCR testing has been around for more than 30 years. but its not a new technology. In fact, PCR tests have beenHow has accuracy and specificity changed?used in clinical diagnostics for more than 30 years. Today,At Mayo Clinic, we have been using PCR routinely in microbiology PCR technology has been refined to improve its accuracy anddiagnostics since the 1990s. Initially, to detect amplified DNA, workflow. Now real-time PCR, also known as quantitative PCRwe would perform gel electrophoresis followed by a Southern (qPCR), can be validated not only for detection but quantification,blot. That worked, but was tedious, and interpreting Southern and a new approach called digital PCR (dPCR) even offers direct,blots can be tricky at times. We performed Southern blot assays absolute quantification of an analyte (Figure 1). So what role willto add specificity, and a little sensitivity, but of course optimizing PCR play in the future, both in and out of the COVID-19 spotlight? specificity was dependent on probe design. In the early years, Clinical Laboratory News spoke to Robin Patel, MD, a professorthere were some laboratories, especially research laboratories, of medicine, professor of microbiology, the Elizabeth P. andthat might have been basing analysis on size of amplified DNA Robert E. Allen Professor of Individualized Medicine, and directoralone, but on the clinical side, we always used Southern blots of the Infectious Diseases Research Laboratory at the Mayo Clinicfor specificity.in Rochester, Minnesota, about how PCR tests have evolved,We transitioned to qPCR around the turn of the century. That how theyre being used right now (with sometimes surprisingchange was advantageous for workflowit is a lot faster and results)and how its everyones job to make sure clinical testingmuch less tedious than running a gel and Southern blot. And is best serving patients. there is a huge improvement for contamination control.Figure 1. Comparison of qPCR and dPCR. (A) A specimen contains a mixture of nucleic acids, as subset of which corresponds to the template of interest (red). (B) In qPCR, a single reaction mixture containing a heterogeneous mixture of template and non-template molecules is prepared. (C) The PCR is subjected to thermocycling, with detection of a fluorescent reporter (intercalating agent or probe) that marks the specific amplification of the target of interest occurring after each cycle. After an end point is reached (D), the relative or absolute concentration of the target of interest is subsequently inferred relative to an internal or external calibrator that is amplified in parallel (E). (F) During digital PCR (dPCR), the specimen is combined with PCR reagents and then subdivided into individual partitions such that on average each contains fewer than a single copy of the template molecule of interest. The array of partitions is subjected to thermal cycling (G) until an end point is reached (H), after which partitions are ascertained for a fluorescent reporter that indicates the successful amplification detected by a (I) camera of the specified target. (J) Quantification of the partitions that are positive vs. negative for amplification allows the absolute concentration of original target molecules to be inferred using Poisson statistics. Adapted from [1] with permission. 7 Molecular testing thermofisher.com/infectiousdisease Contents'