Citation:

Parshionikar, S., J L. Cashdollar, AND G S. Fout. DEVELOPMENT OF MULTIPLEX RT-PCR FOR THE DETECTION OF REOVIRUS, HEPATITIS A VIRUS, POLIOVIRUS, NORWALK VIRUS AND ROTAVIRUS. Presented at Annual Meeting of the American Society for Microbiology, New Orleans, LA, May 23-27, 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:

Water sources are often found to be contaminated by enteric viruses. This is a public health concern as food and waterborne outbreaks caused by enteric viruses such as noroviruses, rotaviruses, hepatitis A virus (HAV) and enteroviruses are a common occurrence. All of these viruses are transmitted by the oral-fecal route. Cell culture detection of these viruses is time consuming and not possible for viruses that cannot be cultured (for example, noroviruses). Reverse transcription-polymerase chain reaction (RT-PCR) assays are commonly used for detection of human enteric viruses in environmental water samples. RT-PCR provides a means to detect low levels of these viruses rapidly. Multiplex RT-PCR is a modification of RT-PCR by which several viruses can be amplified simultaneously in a single reaction using multiple primers pairs. We report the development of multiplex RT-PCR assays to detect reovirus, rotavirus, HAV, poliovirus and Norwalk virus. Primer pairs for all the viruses were designed such that most of the existing strains of each virus could be detected. The method was optimized for Mg+2 concentration, relative primer concentration, template concentration and annealing time and temperature. Mg+2 and relative primer concentration seemed to be the most crucial factors in the success of the assay. The presence of the viruses was confirmed by dot blot hybridizations. The method was shown to be as sensitive as the monoplex RT-PCR for each virus. The method when applied to seeded Ohio River water samples yielded positive results for three viruses. Multiplex RT-PCR format which, when used for detection of viruses in environmental water sources, is a useful tool as it reduces the number of assays needed for each sample thereby facilitating virus detection in a rapid and economical manner.

Record Details: