Citation:

Dean, S., J. Willis, M. Sivaganesan, S. Friedman, AND O. Shanks. Improved Entero1a Quantitative Real-Time PCR Method for Characterization of Enterococcus spp. Levels in Ambient Surface Water Samples. ASM Microbe 2024, Atlanta, GA, June 13 - 17, 2024.

Impact/Purpose:

EPA Method 1609.1 is a quantitative real-time PCR method designed to measure the concentration of Enterococcus spp. in recreational surface waters and has been nationally validated via a multiple laboratory study led by the U.S. EPA Office of Water. This method was the first molecular methodology incorporated in U.S. EPA Recreational Water Quality Criteria for optional use as a beach notification advisory tool, allowing managers to measure water quality and report results to the public on the same day.  While EPA Method 1609.1 was a cutting-edge protocol when first shared with the public in 2015, there have been significant scientific advances in the application of molecular methods for environmental sample testing, providing the opportunity to streamline this methodology to improve precision and reproducibility as well as reduce sample testing time and cost.  In response, the U.S. EPA ORD maintains an active research program to develop, validate, implement, and provide technical support for tools to characterize fecal pollution in recreational waters.  Information covered in this abstract was prepared based on research priorities defined in the U.S. EPA Strategic Research Action Plan (SSWR 403.1.1).

Description:

The use of quantitative real-time PCR (qPCR) to estimate Enterococcus spp. levels allows for same day notification of recreational water quality conditions, representing a major advance over traditional cultured-based methods that require 18 or more hours to obtain results.  The original Entero1a qPCR method utilizes a delta Cq model to estimate Enterococcus spp. concentrations in a water sample, relying on both an Enterococcus faecalis whole cell calibrator and a salmon DNA-based spike control to identify and correct for potential sample matrix interference.  In this study, a streamlined Entero1a qPCR protocol is introduced that eliminates the cell-based calibrator and implements a conditional delta Cq model to estimate Enterococcus spp. concentrations.  Performance of each qPCR approach is compared in head-to-head experiments to investigate the range of quantification, analytical precision, and genetic marker concentration estimates in marine, estuarine, and fresh recreational surface water samples (n = 60).  Findings indicate that the modified Entero1a protocol reduces error in enterococci concentration estimates by up to 78.7% without compromising consistency in mean concentration results (R2 = 0.980; p < 0.0001).  In addition, a new thermal cycling profile is introduced that shortens amplification time by 35.5% and the use of the recently released National Institute of Standards and Technology Standard Reference Material® 2917 is demonstrated for standard curve generation.  The improved Entero1a qPCR protocol should provide more accurate, reproducible, and rapid estimations of fecal contamination for future recreational water quality monitoring applications.

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