title: Rapid detection of infectious rotavirus group A using a molecular beacon assay.
abstract: Rapid, sensitive and specific methods are necessary to detect and quantify infectious viruses. Cultivating and detecting enteric viruses in cell culture are difficult, thus impairing the advancement of knowledge regarding virus-induced diarrhea. Rotavirus (RV) detection has been conducted by serological or molecular biology methods, which do not provide information regarding viral infectivity. Molecular beacons (MBs) have demonstrated efficacy for viral detection in cell culture. We propose a MB assay to detect human rotavirus group A (HuRVA) in cell culture. MA104 cells were mock-infected or infected with HuRVA strains (RotaTeq(®) vaccine and K8 strains), and a specific MB for the HuRVA VP6 gene was used for virus detection. Mock-infected cells showed basal fluorescence, while infected cells exhibited increased fluorescence emission. MB hybridization to the viral mRNA target of HuRVA was confirmed. Fluorescence increased according to the increase in the number of infectious viral particles per cell (MOI 0.5-MOI 1). This technique provides quick and efficient HuRVA detection in cell culture without a need for viral culture for several days or many times until cytopathic effects are visualized. This methodology could be applied in the selection of samples for developing RV vaccines.
authors.affiliation: Department of Genetics, Evolution and Bioagents, Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP 13083-970, Brazil. Electronic address: jessicabertol@hotmail.com.
authors.forename: Jéssica Wildgrube
authors.initials: JW
authors.lastname: Bertol
authors.affiliation: Department of Genetics, Evolution and Bioagents, Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP 13083-970, Brazil. Electronic address: msvgatti@unicamp.br.
authors.forename: Maria Silvia Viccari
authors.initials: MS
authors.lastname: Gatti
mesh_terms: D036022:Capsid Proteins / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D006801:Humans; D015336:Molecular Probe Techniques; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000821:virology; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins
keywords: Cell culture; Molecular beacon; Rotavirus