Citation:

Grimm, A C., J L. Cashdollar, F P. Williams, AND G S. Fout. DEVELOPMENT OF AN ASTROVIRUS RT-PCR DETECTION ASSAY FOR USE WITH CONVENTIONAL, REAL-TIME, AND INTEGRATED CELL CULTURE/RT-PCR. CANADIAN JOURNAL OF MICROBIOLOGY 50(4):269-278, (2004).

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:

Astrovirus is a common cause of gastroenteritis in humans that has been determined to be responsible for outbreaks of illness in several countries. Since astrovirus can be waterborne, it is important to be able to identify this virus in environmental water. We have developed and optimized an RT-PCR method that was able to amplify all eight astrovirus serotypes. In addition, an internal control was designed so that any inhibitors of this astrovirus assay could be detected. The assay was adapted for use in a real-time PCR assay and the sensitivity of these two methods was compared. The real-time assay was then combined with CaCo2 cell culture to produce an integrated cell culture/RT-PCR (ICC/RT-PCR) assay and the sensitivity of the combined assay was compared to RT-PCR alone. The methods were used to detect astrovirus in acute phase illness stool samples as well as to detect the virus in water samples spiked with astrovirus.

Record Details: