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Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR
Citation:
FOUT, G., N. BRINKMAN, J. CASHDOLLAR, S. GRIFFIN, B. MCMINN, E. RHODES, E. VARUGHESE, M. Karim, A. GRIMM, S. A. Spencer, AND M. A. Borchardt. Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. U.S. Environmental Protection Agency, Washington, DC, 2012.
Impact/Purpose:
The overarching objective of this task is to provide Agency scientists and others the methods they need to measure the occurrence of waterborne viral pathogens. The method improvements will facilitate the development of risk-based assessments and tools used by the Agency to set regulations, policies and priorities for protecting human health.
Description:
Version 1.1 - Enteroviruses and noroviruses that may be present in environmental or finished drinking waters are concentrated by 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 extracted from the concentrate and tested for enterovirus and norovirus RNA using real time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve.
URLs/Downloads:
EPA Method 1615Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR (PDF, NA pp, KB, about PDF)