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NIST SRM® 2917 Interlaboratory Performance and Quantitative PCR Testing Benchmarks
Citation:
Willis, J., M. Sivaganesan, J. Kralj, S. Servetas, M. Hunter, S. Jackson, AND O. Shanks. NIST SRM® 2917 Interlaboratory Performance and Quantitative PCR Testing Benchmarks. 21st Symposium on Health-related Water Microbiology WaterMicro2023, Houston, TX, June 15 - 19, 2023.
Impact/Purpose:
National implementation of real-time quantitative PCR recreational water quality monitoring tools requires the development of and access to a high-quality standard control material. This manuscript reports a single laboratory qPCR performance assessment of the National Institute of Science and Technology Standard Reference Material 2917 (NIST SRM® 2917), a linearized plasmid DNA construct that functions with multiple water safety qPCR assays. Findings demonstrated that NIST SRM® 2917 functions with all qPCR methods and suggests that the future use of this control material by scientists and water quality managers should help reduce variability in concentration estimates and make results more consistent between laboratories. This effort addresses an EPA Office of Water high research priority described in the ORD Research Action Plan (SSWR 3.1.1).
Description:
Surface water quality quantitative polymerase chain reaction (qPCR) technologies are expanding from a subject of research to routine environmental and public health laboratory testing. A readily available, reliable standard calibrant material is needed to interpret qPCR measurements, particularly across laboratories. The National Institute of Standards and Technology Standard Reference Material® 2917 (SRM 2917) is a DNA plasmid construct that functions with multiple water quality qPCR assays allowing for the estimation of total faecal pollution and identification of key faecal sources. This study investigates SRM 2917 interlaboratory performance based on repeated measures of 12 qPCR assays by 14 laboratories (n = 1,008 instrument runs). Performance experiments indicate the generation of calibration models (standard curves) with coefficient of determination values (R2) ≥ 0.992. Amplification efficiencies were within expert recommended range of 0.90 to 1.10 for 99.5% (919 of 925) of models with unacceptable values ranging from 0.88 to 0.89. Using a Bayesian approach, a total of 84 single-instrument runs generated across 14 laboratories were used to generate assay-specific global calibration models. Within-lab variability of global calibration model slope and intercept parameters were always less than or equal to between-lab variability, regardless of qPCR assay. Comparable data sets generated by two additional laboratories were then used to assess candidate SRM 2917 data acceptance metrics. Findings indicate that SRM 2917 allows for highly reproducible single-instrument run calibration models and global models offer multiple data acceptance metric options that future users can employ to minimize variability, improve comparability of data across laboratories, and increase confidence in qPCR measurements.
