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DETECTION BY PCR OF HUMAN ENTERIC VIRUSES CONCENTRATED FROM LARGE VOLUMES OF WATER
Citation:
Fout, G S. DETECTION BY PCR OF HUMAN ENTERIC VIRUSES CONCENTRATED FROM LARGE VOLUMES OF WATER. Presented at EPA Workshop for Development of Protocols for Reliable Genetic Methods for Viruses for Use in EPA's Drinking Water Program, Cincinnati, OH, January 15-16, 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:
Viruses are recovered and concentrated from water by passage through a positively charged cartridge filter. Following virus elution from the cartridge filter with beef extract and concentration of the beef extract solution, viruses are usually assayed by cell culture. However, cultural methods are too time consuming and expensive for routine use and many of the viruses that cause waterborne disease are either very difficult to culture or cannot be cultured.
Rapid polymerase chain reaction (PCR) methods have been developed to overcome these problems. While PCR methods are rapid and can detect all the virus groups known to cause waterborne disease, the methods have several unique problems that can cause false negative and false positive results. The presence of potent environmental inhibitors of PCR that are co-concentrated along with viruses during sample processing can result in false negative results. In addition, aerosols containing previously amplified products can cause false-positive results. The quality control measures needed to prevent these problems are much more complex than those used in traditional microbiology or chemistry laboratories.
The number and types of environmental inhibitors vary among different water types and even within a single water type. Thus PCR methods must be designed to remove as many inhibitors as possible from different water types without affecting virus recovery. A multiplex PCR method was developed at the U.S. EPA to measure the occurrence of enteroviruses, reoviruses, rotaviruses, hepatitis A virus and Norwalk virus in water. The method uses a celite-based elution/reconcentration procedure. It results in concentrating a portion of the filter eluate by greater than 800 fold while providing a 74% recovery of poliovirus. Although the method was very effective, false negative and positive results still occur. In one study false negative results were observed in 14% of the samples and false positive results in 6%. The method and appropriate quality controls will be described.