Citation:

Jackson, S., J. Kralj, B. Toman, S. Servetas, M. Hunter, J. Hey, AND O. Shanks. A Plasmid DNA Standard for Molecular Recreational Water Quality Testing. DHS/NIST Standards to Support an Enduring Capability in Wastewater Surveillance for Public Health, Virtual, June 14 - 18, 2021.

Impact/Purpose:

EPA and other scientists have made significant strides in the development of advanced monitoring technologies for the characterization of fecal pollution.  Genetic technologies are now available that can provide same day notification of fecal pollution levels using enterococci and E. coli, as well as provide fecal pollution information for human, ruminant, canine, and avian sources in recreational water samples.  These methodologies rely on real-time quantitative PCR (qPCR) to estimate the concentration of genetic targets using a standard curve generated from a reference material.  National implementation of a qPCR-based technologies requires a high-quality reference material that is readily available to the public.  This project sought to develop a national standard reference material to function with 13 qPCR surface water quality testing methods in collaboration with the National Institute of Standards and Technology (NIST).  The resulting material, NIST SRM 2719 will help federal, state, and other local groups implement these advanced water quality testing technologies on a more consistent basis.

Description:

Contamination of waterways with fecal material can lead to the dissemination of pathogens that can have serious impacts on human and environmental health. The U.S. Environmental Protection Agency (EPA) is responsible for protecting human and environmental health through the safeguard of recreational waters. Traditional monitoring practices involve time consuming cultivation procedures which can delay decision making. To accelerate this process molecular techniques, specifically quantitative polymerase chain reaction (qPCR) methods, for identifying host-associated genetic markers of fecal pollution and quantifying fecal indicator bacteria have been developed at EPA and by other researchers. National implementation of these methods requires rigorous proficiency testing by state and local public health agencies, as well as commercial entities that hope to adopt these methods.  In a collaborative effort to support the adoption of these molecular methods, the EPA and NIST developed a plasmid DNA Standard Reference Material (NIST SRM 2917) for the purpose of implementing select qPCR assays for recreational water quality monitoring applications. The material consists of 6 Levels of a linearized plasmid DNA containing 13 single-copy PCR targets selected by the EPA.  The vector consists of a standard pUC plasmid with an ampicillin resistance gene, an origin of replication, and M13 universal priming sites flanking the target genetic marker construct. Dilution levels span approximately 5 to 500,000 plasmid copies per ¿L. Each tube of material contains approximately 200 ¿L of plasmid DNA in TE buffer (pH 8.0) with 10 ng/¿L of RNA stabilizer (yeast tRNA) in a 1.5 mL low retention microcentrifuge vial.  The material is stored at 4 °C and should not be frozen. Approximately 1,000 units were generated. The material was characterized by NIST using droplet digital PCR to establish homogeneity and stability of the material as well as assign an absolute copy number to each dilution level. 

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