Citation:

Sivaganesan, Mano, M. Varma, S. Siefring, AND Rich Haugland. Quantification of plasmid DNA standards for U.S. EPA fecal indicator bacteria qPCR methods by droplet digital PCR analysis. JOURNAL OF MICROBIOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 152:135-142, (2018). https://doi.org/10.1016/j.mimet.2018.07.005

Impact/Purpose:

A previous obstacle to establishing widely useful standardized data acceptance criteria for U.S. Environmental Protection Agency (EPA) qPCR methods has been the unavailability of standardized reference materials. This report describes the application of droplet digital PCR (ddPCR) analysis for the quantification of a set of synthetic, plasmidderived DNA standards that can be used in EPA qPCR methods for analysis of enterococci and Escherichia coli fecal indicator bacteria in recreational waters. EPA has provided these standards to laboratories across the country and most recently for a multi-laboratory validation study of EPA Draft Method C for E. coli

Description:

An obstacle to establishing widely useful data acceptance criteria for U.S. Environmental Protection Agency (EPA) qPCR methods has been the unavailability of standardized reference materials. Earlier versions of EPA Methods 1609 and 1611 for enterococci used cellular reference materials for quantifying enterococci in unknown test samples, however, EPA updates to these fundamentally DNA-based analysis methods have shifted toward the use of DNA standards. This report describes the application of droplet digital PCR (ddPCR) analysis for the quantification of a set of synthetic plasmid DNA standards that have been made available for updated EPA Methods 1609.1 and 1611.1 as well as for EPA Draft Method C for Escherichia coli. To obtain the most accurate concentration estimates possible, part of this effort was to develop a data analysis model for determining the fluorescence thresholds that distinguish positive from negative droplets produced by the ddPCR reactions. Versions of this model are described for applications with individual reactions, multiple reactions within a ddPCR system run, and multiple reactions within and across different system runs. The latter version was applied toward determinations of error in the concentration estimates of the standards from replicate analyses of each standard in multiple ddPCR system runs. Mean concentration estimates for the five standards from the ddPCR analyses were 4.356, 3.381, 2.371, 1.641 and 1.071 log10 copies/5 μL with associated standard deviations of 0.074, 0.082, 0.108, 0.131 and 0.188, respectively. These estimates contrasted with expected log10 concentrations of 4.6, 3.6, 2.6, 1.9 and 1.3 copies/5 μL, respectively, based on the yield of the plasmid reported by the vendor and spectrophotometric analysis of the initial stock solution of this material. These results illustrate how the analyses of original stocks may lead to potential bias(es) in the concentration estimates of final DNA standards and subsequently in the estimates of unknown test samples determined from these standards in qPCR analyses.

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