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AN INTEGRATED CELL CULTURE/RT-PCR METHOD FOR DETECTING ENTEROVIRUS IN WATER
Citation:
VILLEGAS, L. F. AND G. FOUT. AN INTEGRATED CELL CULTURE/RT-PCR METHOD FOR DETECTING ENTEROVIRUS IN WATER. Presented at American Society for Microbiology 106th General Meeting, Orlando, FL, May 21 - 25, 2006.
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 2 to "develop new data, innovative tools and improved technologies to support decision making by the Office of Water on the Contaminant Candidate List and other regulatory issues" 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 method improvements will facilitate the development 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 Objectives
Subtask A: Improving sample collecting, virus concentration and sample preparation
o Develop a less expensive alternative to the Virosorb 1 MDS filter.
Subtask B: Molecular and Cultural Assays
o Investigate methods to improve the reverse transcription step in reverse transcription-polymerase chain reaction (RT-PCR) assays by when, for what purpose, for what client.
o Development of complete real-time assays that can be used for screening environmental samples.
o Develop a reporter gene cell culture system to indicate if virus infection has occurred without the use of molecular assays.
o Use improved cell culture lines to develop assays for nonculturable or poorly growing viruses.
Description:
Echovirus and coxsackievirus can cause mild to severe disease following consumption of contaminated drinking water. However, comprehensive occurrence studies of enteroviruses in drinking water matrices are limited, in part because of the lack of available methods that are rapid, sensitive, and able to detect infectious virus. To address this issue, the US EPA has included echoviruses and coxsackieviruses on the microbial contaminant candidate list (CCL). To provide the research needed to support regulatory decisions for these CCL viruses, our efforts have focused on developing methods for the rapid detection of infectious virus. It has become increasingly important to design a method which can detect infectious virus in a rapid and sensitive matter. To achieve this goal, we have developed a technique which integrates cell culture and molecular assays into one method. This integrated cell culture/reverse transcription polymerase chain reaction method (ICC/RT-PCR) approach uses molecular tools to rapidly detect infectious enterovirus in water matrices.