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THE USE OF RT-PCR FOR THE DETECTION OF ENTERIC VIRUSES IN PRAIRIE SURFACE DRINKING WATER SUPPLIES
Citation:
Ruecker, N. J., G S. Fout, H. G. Peterson, S. Watson, J. Lawrence, G. Appleyard, AND N. Christofi. THE USE OF RT-PCR FOR THE DETECTION OF ENTERIC VIRUSES IN PRAIRIE SURFACE DRINKING WATER SUPPLIES. Presented at 37th central Canadian Symposium on Water Pollution Research, Burlington, Ontario, February 4-5, 2002.
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:
Concerns over the microbial safety of drinking water supplies have focused on bacteria and parasites while the occurrence of pathogenic waterborne viruses have been largely ignored. In fact, water supplies are not routinely monitored for human enteric viruses. This is despite the fact that pathogenic viruses are estimated to acccount for more than half of waterborne diseases. Reverse transcriptase polymerase chain reaction (RT-PCR) is used widely to detect viruses in clinical samples, but the application of RT-PCR to source-waters is compounded by two major factors. Relative to clinical samples, these water contain low numbers of pathogenic viruses and contain substances which are inhibitory to RT-PCR reactions. These substances inevitably become concentrated along with the virus particles. Without purification of the target viruses to remove inhibitors, false negatives are likely to occur, with serious health implications. We tested an RT-PCR method by screening four prairie surface water supplies for the presence of major enteric viruses (enterovirus, reovirus, rotavirus, hepatitis A virus and calicivirus). At the same time we tested for the presence of other microbes, and measured a range of water quality parameters. The presence of coliform bacteria, high particle and turbidity levels as well as dissolved inorganic and organic material indicate that these source waters were poor quality. No viruses were detected in the samples. However, the same samples seeded with virus also resulted in a few confirmed positive results. We argue that there is clearly a critical need to develop methods which are robust, that can be used to detect waterborne viruses with confidence, particularly for waters of poor quality. This is particularly important as global population density, intensive livestock farming and water usage increase.