title: Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure.
abstract: A method was developed for discriminating damaged viruses or naked viral RNA from intact viruses by ethidium monoazide (EMA) treatment before RT-PCR. The applied EMA treatment consisted of three steps: (1) EMA dose, (2) exposure to light, and (3) additional purification by spin-column gel filtration. Approximately 4-log reduction in viral RNA concentration was observed by adding a dose of 10 μg/mL-EMA with 300 s of light irradiation. Although residual EMA can be an inhibitor of RT-PCR, its effect was reduced by spin-column gel filtration or a QIAamp® Viral RNA Mini Kit. EMA-RT-PCR was applied to the thermally treated PV1. Results of EMA-RT-PCR were similar to the plaque assay when PV1 was thermally inactivated. Although this is a preliminary study investigating applicability of the EMA-RT-PCR method for RNA viruses, the results suggest that the method is potentially applicable for the selective detection of epidemiologically important enteric viruses in water such as enteroviruses and noroviruses.
authors.affiliation: Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. kim-kyungju@metawater.co.jp
authors.forename: K
authors.initials: K
authors.lastname: Kim
authors.forename: H
authors.initials: H
authors.lastname: Katayama
authors.forename: M
authors.initials: M
authors.lastname: Kitajima
authors.forename: Y
authors.initials: Y
authors.lastname: Tohya
authors.forename: S
authors.initials: S
authors.lastname: Ohgaki
mesh_terms: D000818:Animals; D001386:Azides / Q000494:pharmacology*; D006358:Hot Temperature; D008027:Light; D051379:Mice; D029322:Norovirus / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000528:radiation effects; D012328:RNA Viruses / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification / Q000528:radiation effects; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001386:Azides; D012367:RNA, Viral; C014535:8-azidoethidium