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Ohio Water Microbiology Lab

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Quality Assurance/Quality Control Manual: Ohio Water Microbiology Laboratory

Methods for enteric viruses
(Note: Appendices for enteric viruses are available upon request.)

Samples for enteric viruses are analyzed by the OWML by use of USEPA Method 1615 (USEPA, 2012b). Method 1615 includes two methods for analyzing water samples — reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and cell culture. The RT-qPCR method quantifies human enteroviruses and human noroviruses by targeting genetic sequences specific to these viruses. Molecular methods, such as RT-qPCR, do not determine infectivity of viruses, but they have an advantage in that they can detect many more types of enteric viruses. The cell culture method detects enterovirus and orthoreovirus species that are capable of infecting and producing cytopathic effects (CPE) in the Buffalo Green Monkey Kidney (BGM) cell line. Not all viruses can be detected by cell culture, including noroviruses. Cell culture is used to determine whether the enteroviruses are infectious.

Protocols for RT-qPCR can be found in Appendix E1 (elution and organic flocculation protocol), Appendix E2 (foam elution and concentration), and Appendix E3 (reverse transcription and qPCR protocol). The sample processing benchsheets can be found in Appendixes E1a and E2a, and the qPCR batch sheets can be found in Appendix E1b. Appendix E3a contains the assay specific qPCR benchsheets with detailed information about each assay. The cell culture protocol can be found in Appendix E4, along with bench sheets for cell culture and viral titer results (Appendix E4a). The method for collection and initial processing of water samples for human polyomavirus and the corresponding bench sheet are found in Appendixes E5 and E5a. Extraction for human polyomavirus is described in Appendix E6.



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