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Method 1615 Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR
Fout, Shay, N. Brinkman, J. Cashdollar, S. Griffin, B. McMinn, E. Rhodes, E. Varughese, M. Karim, A. Grimm, S. Spencer, AND M. Borchardt. Method 1615 Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. U.S. Environmental Protection Agency, Washington, DC, 2014.
EPA Method 1615 provides regulatory agencies a tool to determine risk from enteroviruses and noroviruses in drinking waters. The tool is currently being used by EPA under the Unregulated Contaminant Monitoring Rule monitoring round 3 and by several national laboratories. It provides for standardization whereby results from separate analytical laboratories can be compared.
Viruses that may be present in environmental or finished drinking waters are concentrated by passage through an electropositive filter. Viruses are eluted from the filter 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 test 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 further concentrated by centrifugal ultrafiltration. The viral ribonucleic acid (RNA) is extracted from the concentrate and tested for enterovirus and norovirus RNA using 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.