Principles of Real-Time PCR
Pp. 3-17 (15)
Amanda D. Loftis and Will K. Reeves
Compared with traditional PCR assays, diagnostic assays based upon real-time PCR technology
have increased speed and dynamic range; in addition, they enable quantitative analysis of gene copies and
have the potential for increased specificity when nucleic acid probes are used. Optimized real-time PCR
assays can also be highly sensitive, detecting as few as 1-10 copies of a target gene in a nucleic acid sample.
Adopting real-time PCR in a diagnostic laboratory requires an understanding of these assays, including both
the benefits and drawbacks unique to this technology. An overview of real time PCR applications is
presented here, with an emphasis on practical issues that might affect implementation of real-time PCR
testing in a diagnostic laboratory. Increased cleanliness and process controls are required in the laboratory,
to prevent contamination of sensitive real-time PCR. Nucleic acid extraction procedures, using one of the
many available chemistries, should be carefully optimized for reproducible, efficient extraction of nucleic
acids that are free of PCR inhibitors. Reverse transcription of RNA adds an additional variable that can
affect quantitative data. For the assay itself, different options have been developed for the detection of
products in real-time, including dye-based assays, hydrolysis probes, and hybridization probes. Different
options and the benefits and drawbacks of each are discussed. Finally, specific applications for real-time
quantitative PCR assays in diagnostic laboratories are highlighted.
Real-time PCR; polymerase chain reaction; clinical laboratory techniques; reverse
transcription; DNA; RNA; nucleic acid probes.
Ross University School of Veterinary Medicine, P.O. Box 334, Basseterre, St. Kitts, St. Kitts and Nevis.