Citation:

FOUT, G. QUALITY ASSURANCE FOR PCR. Presented at Major Accomplishments and Future Directions in Public Health Microbiology Workshop, Columbus, OH, February 15 - 18, 2005.

Impact/Purpose:

Overarching Objectives and Links to Multi-Year Planning

This task directly supports the Drinking Water Research Program Multi-Year Plan's long term goal to "develop scientifically sound data and approaches to characterize and manage risks to human health posed by exposure to waterborne pathogens and chemicals" under GRPA Goal 2 (Clean and Safe Water). The overarching objective is to provide the Office of Water, Agency risk assessors and managers, academics, the scientific community, state regulators, water industry and industry spokes groups with exploratory occurrence and exposure data on human enteric viruses. These data will improve the quality of risk-based assessments and tools used by the Agency to set regulations, policies and priorities for protecting human health and allow the Agency to assure the public that the appropriate methods are being used to demonstrate that drinking water is safe from pathogenic agents.

Specific Subtask Objectives:

o Conduct an exploratory occurrence studies on emerging human waterborne pathogenic viruses and viruses on the Contaminant Candidate List (CCL) in water (Subtask A; to be completed by 9/05 in support of LTG 1 (due 2010)).

o Determine the relationship of bacterial virus indicators to human enteric virus occurrence in the above studies (Subtask A; to be completed by 9/05 in support of LTG 1 (due 2010)).

o Develop a non-invasive assay for measuring human exposure to viruses (Subtask B; to be completed by 9/05 in support of LTG 1 (due 2010)).

Description:

The U.S. Environmental Protection Agency (EPA) held a workshop in January 2003 on the detection of viruses in water using polymerase chain reaction (PCR)-based methods. Speakers were asked to address a series of specific questions, including whether a single standard method could be developed and used for regulatory purposes. Key conclusions of the workshop were that 1) there will be an increasing need for PCR and other molecular methods in the analyses of environmental samples, especially for agents of regulatory importance which cannot be cultured, but 2) the lack of standardization of QA/QC practices reduces the value of PCR-based data. As a result of these conclusions, EPA's Office of Water and Office of Research and Development developed and published a guidance document in October 2004 on quality assurance procedures entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples" (EPA publication number EPA 815-B-04-001). This presentation will present an overview of this guidance, focusing on the key areas facility design, workflow and quality controls. The guidance recommends that PCR facilities use at least three laboratories: 1) a reagent preparation room, 2) a sample preparation room and 3) an amplification and product room. The characteristics of each room will be described. Workflow should proceed from the reagent preparation room to the sample preparation room to the amplification and product room, and never in reverse. It is recommended that four types of positive and two types of negative controls be routinely used. These consist of a "PCR Positive Control," which is used to verify that reagents have been prepared properly; an "Inhibition Positive Control," which is used to show that interfering constituents of the environmental matrix have been removed adequately and do not interfere with the PCR portion of the method; a "Method Positive Control," which is used to demonstrate that the entire method (sampling, concentration, inhibitor removal, PCR, confirmation) is working; and a "Matrix Spike," which gives confidence that the matrix is not interfering with recovery and detection of target organisms over the entire method. The negative controls consist of a "PCR Negative Control," which shows that the PCR reagents do not contain contaminating substances; and a "Method Blank," which verifies that contamination has not been introduced throughout the processing and assay steps. Procedures for reducing QC failures and for taking corrective actions also will be described.

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