Citation:

Sivaganesan, M., J. Willis, M. Karim, A. Babatola, D. Catoe, A. Boehm, M. Wilder, H. Green, A. Lobos, V. Harwood, S. Hertel, Rich Haugland, R. Klepikow, M. Howard, P. Laksanalamai, A. Roundtree, M. Mattioli, S. Eytcheson, M. Molina, M. Lane, R. Rediske, A. Ronan, N. D’Souza, J. Rose, A. Shrestha, C. Hoar, A. Silverman, W. Faulkner, K. Wickman, J. Kralj, S. Servetas, M. Hunter, S. Jackson, AND O. Shanks. NIST SRM® 2917 Interlaboratory Performance and Quantitative PCR Performance Benchmarks. ASM Microbe, Houston, TX, June 04 - 09, 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. Abstract 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 403.1.1).

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. Numerous qPCR methodologies are available to assess surface water safety for recreational activities and identify specific pollution sources. The precision and accuracy of qPCR measurements are strongly influenced by the quality and reproducibility of the calibration model. Therefore, a readily available, reliable reference material is needed to interpret qPCR measurements, particularly across laboratories. Standard Reference Material® 2917 (NIST SRM® 2917) is a DNA plasmid construct that functions with multiple water quality qPCR assays allowing for the estimation of total fecal pollution and identification of key fecal sources including human, cattle, canine, ruminant, swine, avian, and general fecal indicator bacteria. This study investigates SRM 2917 interlaboratory performance based on repeated measures of 12 qPCR assays by 14 laboratories (n = 1008 instrument runs). Using a Bayesian approach, the performance of each single-instrument run is evaluated and then data are combined to generate assay-specific global calibration models allowing for characterization of within- and between-lab variability. Finally, a series of performance criteria are proposed for future users. Performance experiments indicate all qPCR single-instrument run calibration models exhibit a high degree of linearity with R2 ≥ 0.992 and efficiency where 99.5% fell within the recommended 0.90 to 1.10 range indicating that SRM 2917 allows for reproducible single-instrument run calibration models across laboratories, regardless of qPCR assay. Comparable data sets generated by two additional laboratories are then used to assess candidate SRM 2917 data acceptance metrics. Results show that global model data acceptance metrics offer multiple options to minimize variability, improve comparability of data across laboratories, and increase confidence in qPCR measurements. This assessment represents a necessary step towards large scale method adoption not only in the water quality testing arena, but also in the public acceptance of these technologies.

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