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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVEMENT OF DAIRY FORAGE AND MANURE MANAGEMENT TO REDUCE ENVIRONMENTAL RISK Title: Method 1615 measurment of Enterovirus and Norovirus occurrence in water by culture and qRT-PCR

Authors
item Fout, G -
item Brinkman, N -
item Cashdollar, J -
item Griffin, S -
item Mcminn, B -
item Rhodes, E -
item Varughese, E -
item Grimm, A -
item Spencer, Susan
item Borchardt, Mark

Submitted to: Government Publication/Report
Publication Type: Government Publication
Publication Acceptance Date: June 14, 2011
Publication Date: December 1, 2011
Citation: Fout, G.S., Brinkman, N.E., Cashdollar, J.L., Griffin, S.M., Mcminn, B.R., Rhodes, E.R., Varughese, E.A., Grimm, A.G., Spencer, S.K., Borchardt, M.A. 2011. Method 1615 measurment of Enterovirus and Norovirus occurrence in water by culture and qRT-PCR. Government Publication/Report. EPA/600/R-10/181.

Technical Abstract: Viruses that may be present in environmental or finished drinking waters are concentrated from passage through electropositive filters. Viruses are eluted from the filters with a beef extract reagent and concentrated using organic flocculation. A portion of the concentrated eluate is then inoculated onto replicate flasks of BGM cells to measure infectious viruses. Cultures are examined for the development of cytopathic effects for two weeks and then re-passaged onto fresh cultures for confirmation. Virus concentration in each sample is calculated in terms of the most probable number (MPN) of infectious units per liter using EPA’s MPN calculator. For molecular assays, the concentrated eluate is concentrated again by centrifugal ultrafiltration. The RNA is then extracted from the concentrate and tested for enterovirus and norovirus RNA using real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve.

Last Modified: 12/22/2014
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