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DEVELOPMENT OF A MOLECULAR METHOD TO IDENTIFY ASTROVIRUS IN WATER.
Grimm, A C., J L. Cashdollar, F P. Williams, AND G S. Fout. DEVELOPMENT OF A MOLECULAR METHOD TO IDENTIFY ASTROVIRUS IN WATER. Presented at Science and Mission Club, Cincinnati, OH, June 4, 2003.
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)
Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several outbreaks. Since astrovirus can be waterborne, there is interest in testing environmental water for astrovirus and we have developed a sensitive RT-PCR assay that is designed to detect all known astrovirus. When tested, this assay was able to detect strains from all eight serotypes. In addition, an internal control was developed so that it will be possible to determine if the sample being tested contains PCR inhibitors. Most probable number analysis determined that when amplified with the developed assay, a single DNA molecule of the internal control could be detected if inhibitors were not present. The assay was successfully adapted to real-time PCR and this method was then used for integrated cell culture RT-PCR detection of infectious virus. The methods were successfully used to detect astrovirus present in clinical samples and spiked water samples.