qPCR: Basic Principles and Range of Applications

Oral Presentation

Prepared by R. Noble, A. Blackwood
UNC Chapel Hill Institute of Marine Sciences, 3431 Arendell Street, Morehead City, NC, 28557, United States

Contact Information: rtnoble@email.unc.edu; 252-726-6841


Rapid, cost-effective, and user-friendly molecular diagnostic approaches such as qPCR are poised to complement and in some cases, replace slower culture-based analyses in the arenas of clinical diagnostics, water quality, and food security. For some areas, such as meat and seafood safety, the revolution in molecular diagnostics has already taken place. For water quality monitoring, certain qPCR methods have been approved (e.g. EPA Methods 1609 and 1611). While these methods are not yet widely implemented for water quality monitoring, their use is increasing nationwide. There is global demand for environmental monitoring via qPCR, which features the accurate quantification of fecal indicator bacteria and viral and bacterial pathogens in both water and seafood in hours, rather than days. Because rapid qPCR methods are faster, water quality managers can more accurately protect public health and prevent costly outbreaks. However, many scientists and technicians in water quality microbiology laboratories have limited experience using these molecular tests. To ensure that practitioners understand and can consider the successful implementation qPCR techniques, a review of qPCR principles and applications will be presented. The presentation will include the main attributes of qPCR assays for fecal indicator bacteria as applied broadly, but will also consider and present the most important attributes related to QA/QC for water quality monitoring including complex matrices such as stormwater, wastewater, and groundwater. A globally-accepted set of minimum guidelines for the application of qPCR approaches will be presented, as well as discussion of absolute and relative quantification, qPCR efficiency, sensitivity, specificity, and reproducibility. Changes in the current regulatory frameworks for qPCR as applied to water quality monitoring will be discussed. Attendees will gain sufficient knowledge of qPCR, including an appreciation for the increasingly user-friendly and inexpensive attributes of qPCR.