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DEVELOPMENT OF VIRAL INTERNAL CONTROLS FOR THE ASSESSMENT OF INHIBITOR REMOVAL FROM ENVIRONMENTAL WATER SAMPLES.
Citation:
Parshionikar, S. AND G S. Fout. DEVELOPMENT OF VIRAL INTERNAL CONTROLS FOR THE ASSESSMENT OF INHIBITOR REMOVAL FROM ENVIRONMENTAL WATER SAMPLES. Presented at 21st Annual Meeting of the American Society for Virology, Salt Lake City, UT, May 19-23, 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:
The presence of human enteric viruses in drinking water is of public health concern. It is therefore important to be able to reliably detect these viruses in contaminating water sources. Cell culture methods are time consuming and also not all enteric viruses can currently be propagated in cell culture, e.g., caliciviruses. Molecular methods of detection such as RT-PCR are rapid and accurate but inhibitors present in environmental water samples can sometimes limit RT-PCR. For this purposel, amplification of internal controls for these viruses can be used to determine efficient removal of inhibitors from environmental samples. The efficiency of PCR is dependent on the length of the region to be amplified and the GC content. Since amplification obtained from an internal control that is significantly different in length and GC content may not be an accurate representation of PCR eficiency, we have developed internal controls for poliovirusus, HAV, Norwalk virus and rotavirus. These internal conrols have deletions of only a few basepairs compared to their wildtype sequences. Each viral internal control was developed such that it had the same primer binding sites as the virus. The probe region for the virus was modified with a new sequence. In particular, a deletion of a small number of viral bases was created and new bases were introduced keeping the GC content as close to the viral genome as possible. Each internal control RT-PCR band was only marginally smaller in length than its viral counterpart. Specifically, poliovirus was 9 bp, HAV was 15 bp, Norwalk was 8 bp and rotavirus was 6 bpsmaller than their respective viral RT-PCR fragments. The internal control RT-PCR bands could be separated from the viral RT-PCR bands by polyacrylamide gel electrophoresis. Each of the internal control DNA was sequenced to confirm the accuracy of the manipulated region. Results of hybridization experiments indicated that the internal control probes were specific and did not cross react with the viral probes and vice-versa. Thus, we have developed internal controls that can be used for assessing inhibitor removal from environmental samples.