Applications of Digital PCR for Clinical Microbiology

摘要

Since its invention in the early 1980s, PCR has become an indispensable tool for the detection of microbiologic agents. With subsequent improvements, such as thermostable polymerases and dedicated instruments, PCR became broadly available to clinical microbiology laboratories (1). The clinical utility of PCR expanded with the development of quantitative PCR (qPCR), which enabled not only detection but also quantification of targeted nucleic acids in clinical specimens. In qPCR, the inclusion of fluorescent DNA-intercalating dyes or fluorescent dye-labeled probes within the PCR allows continuous monitoring of the amplification reaction (real-time PCR) (Fig. 1A). By measuring the PCR cycle at which fluorescence reaches a certain threshold (the threshold cycle [CT] value) for samples with a known amount of target, a standard curve can be generated (Fig. 1B). By comparing the CT of a clinical specimen to the standard curve, the quantity of the analyte can be calculated. The ability to quantify pathogens has proven useful as a prognostic indicator and to monitor treatment response in many infections (2).