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AN ALTERNATIVE ELUENT TO BEEF EXTRACT FOR ELUTING POLIOVIRUS FROM ELECTROPOSITIVE FILTERS
Citation:
Cashdollar, J L., D R. Dahling, AND G S. Fout. AN ALTERNATIVE ELUENT TO BEEF EXTRACT FOR ELUTING POLIOVIRUS FROM ELECTROPOSITIVE FILTERS. Presented at 101st General Meeting of the American Society for Microbiology, Orlando, FL, May 20-24, 2001.
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:
Traditional methods for enteric virus removal from waters involve filtering the water through a positively charged filter followed by elution with beef extract, second step concentration by flocculation, and assay in cell culture. Two of the problems associated with this method are closely related. First, many enteric viruses are non-culturable, and thus are not detected by traditional cell culture methods. Alternative molecular procedures such as those based upon the polymerase chain reaction (PCR) are often used to detect these viruses. Second, it has been reported that beef extract may directly inhibit and concentrate inhibitors of the molecular procedures. This study describes the development of a method which substitutes amino acides for beef extract to elute poliovirus from 1MDS electropositive filters and the subsequent analysis by cell culture and RT-PCR. Initial experiments examined the ability of several different amino acides to elute poliovirus from 47 mm electropositive filters. From there, a concentratoin technique was developed using aluminum sulfate. To test the procedure, 20 L volumes of Ohio River water were seeded with poliovirus and eluted by beef extract, a single amino acid, or a combination of amino acids. Results based upon the plaque assay procedures indicate that alanine, serine, and aminobenzoic acid can elute virus, although poliovirus does not enumerate to the same levels in aminobenzoic acid as beef extract. Poliovirus was also detected in samples eluted by both beef extract and amino acids. In addition, we have been unable to detect any inhibitory RT-PCR reactions with beef extract. Further research is necessary to study viral enumeratoin in aminobenzoic acid and to evaluate the method using larger sample volumes and other enteric viruses.