EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR.
| Autor: | Fout GS; National Exposure Research Laboratory, U.S. Environmental Protection Agency; fout.shay@epa.gov., Cashdollar JL; National Exposure Research Laboratory, U.S. Environmental Protection Agency., Griffin SM; National Exposure Research Laboratory, U.S. Environmental Protection Agency., Brinkman NE; National Exposure Research Laboratory, U.S. Environmental Protection Agency., Varughese EA; National Exposure Research Laboratory, U.S. Environmental Protection Agency., Parshionikar SU; Technical Services Center, Office of Ground Water and Drinking Water, U.S. Environmental Protection Agency. |
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| Jazyk: | angličtina |
| Zdroj: | Journal of visualized experiments : JoVE [J Vis Exp] 2016 Jan 16 (107), pp. e52646. Date of Electronic Publication: 2016 Jan 16. |
| DOI: | 10.3791/52646 |
| Abstrakt: | EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (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. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water. |
| Databáze: | MEDLINE |
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