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QUALITY CONTROLS FOR PCR
Citation:
Fout, G S. QUALITY CONTROLS FOR PCR. Presented at Water Quality Technology Conference, Philadelphia, PA, November 2-5, 2003.
Impact/Purpose:
Overarching Objectives and Links to Multi-Year Planning
This task directly supports the 2003 Drinking Water Research Program Multi-Year Plan's long term goal 1 for "regulated contaminants" and long term goal 2 for "unregulated contaminants and innovative approaches" 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 the methods they need to measure occurrence of waterborne viral pathogens. The methods developed 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 will 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 Evaluate techniques for enhancement of growth of human enteric viruses in support of CCL #2 and #3 and for use in the UCMR (Subtask A; to be completed by 9/05 in support of LTG 2)
o Develop a multiplex RT-PCR method that incorporates internal controls for use in the UCMR (Subtask B; completed 9/03 in support of LTG 2)
o Develop and evaluate new molecular technologies for use in the UCMR. Included will be real-time RT-PCR methods for Norwalk virus and astroviruses, and integrated cell culture/molecular procedures for detection of infectious viruses (Subtask B; to be completed by 9/05 in support of LTG 2)
Description:
The purpose of this presentation is to present an overview of the quality control (QC) sections of a draft EPA document entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples." This document has been prepared by the Office of Water and the Office of Research and Development to serve as guidance for environmental sample analyses involving PCR techniques, for incorporation into new EPA PCR-based methods, for use with EPA grants and assistance agreements involving PCR methods and as guidance to evaluate PCR data in technical papers. This presentation will describe when and how to use four positive controls and two negative controls with PCR methods and will describe the proper controls for assays used to confirm PCR results. The four positive controls consist of a "PCR Positive Control," which is used to verify that reagents have been prepared properly; the "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; the "Method Positive Control," which is used to demonstrate that the entire method (sampling, concentration, inhibitor removal, PCR, confirmation) is working; and the "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 corrective actions will be described.