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Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus
Karim, M., Shay Fout, C. Johnson, K. White, AND S. Parshionikar. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus. JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 219(7):51-61, (2015).
PCR is used to detect many important waterborne viral pathogens because results can be obtained quickly compared to culturing and because many of these viruses cannot be cultured. The problem with PCR is that it does not distinguish between infectious and non-infectious virus particles, and thus the risk from positive findings is uncertain. This paper provides a procedure to allow PCR to be used for measuring only infectious particles for certain virus types. With these types a positive result does have public health implications.
Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay.
VIRMET-D-13-00285R1ACCEPT.PDF (PDF,NA pp, 788.835 KB, about PDF)
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL EXPOSURE RESEARCH LABORATORY
MICROBIOLOGICAL AND CHEMICAL EXPOSURE ASSESSMENT DIVISION
BIOHAZARD ASSESSMENT RESEARCH BRANCH