title: A multiplex RT-PCR assay for the detection of fish picornaviruses.
abstract: With the emergence of high profile fish diseases in the Great Lakes region, surveillance and regulatory inspections of fish populations have increased. This has resulted in a better understanding of known pathogens and isolation of many new pathogens of fish. In this study, a multiplex RT-PCR assay was developed for the detection of three newly discovered fish picornaviruses: bluegill picornavirus-1 (BGPV-1), fathead minnow picornavirus (FHMPV), and eel picornavirus-1 (EPV-1). This assay was found to be very sensitive with a detection limit of 81.9pg/μl of extracted RNA from a pool of FHMPV and BGPV-1 and was able to detect 501 and 224 gene copies/μl of BGPV-1 and FHMPV, respectively. The assay was highly reproducible and did not cross react with other closely related pathogens. We believe that this new assay provides a rapid and cost effective tool for confirming cell culture isolates and conducting prevalence studies of these newly detected fish picornaviruses.
authors.affiliation: Minnesota Veterinary Diagnostic Laboratory, Department of Veterinary Population Medicine, University of Minnesota, 1333 Gortner Avenue Street, Paul, MN 55108, USA. Electronic address: kumars@umn.edu.
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Mor
authors.affiliation: Minnesota Veterinary Diagnostic Laboratory, Department of Veterinary Population Medicine, University of Minnesota, 1333 Gortner Avenue Street, Paul, MN 55108, USA.
authors.forename: Nicholas B D
authors.initials: NB
authors.lastname: Phelps
authors.affiliation: Department of Microbiology, University of Wisconsin-La Crosse, 1725 State Street, La Crosse, WI 54601, USA.
authors.forename: Marisa
authors.initials: M
authors.lastname: Barbknecht
authors.affiliation: Department of Microbiology, University of Wisconsin-La Crosse, 1725 State Street, La Crosse, WI 54601, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hoffman
authors.affiliation: Minnesota Veterinary Diagnostic Laboratory, Department of Veterinary Population Medicine, University of Minnesota, 1333 Gortner Avenue Street, Paul, MN 55108, USA.
authors.forename: Sagar M
authors.initials: SM
authors.lastname: Goyal
mesh_terms: D000818:Animals; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D006114:Great Lakes Region; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010849:Picornaviridae / Q000235:genetics / Q000302:isolation & purification*; D010850:Picornaviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods*
keywords: Bluegill picornavirus; Eel picornavirus; Fathead minnow picornavirus; Multiplex RT-PCR; Reproducibility; Sensitivity; Specificity