A multiplex RT-PCR assay for the detection of fish picornaviruses

Sunil K Mor; Nicholas B D Phelps; Marisa Barbknecht; Michael A Hoffman; Sagar M Goyal

doi:10.1016/j.jviromet.2015.04.025

A multiplex RT-PCR assay for the detection of fish picornaviruses

J Virol Methods. 2015 Sep 1:221:131-4. doi: 10.1016/j.jviromet.2015.04.025. Epub 2015 May 9.

Authors

Sunil K Mor¹, Nicholas B D Phelps², Marisa Barbknecht³, Michael A Hoffman³, Sagar M Goyal²

Affiliations

¹ 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.
² Minnesota Veterinary Diagnostic Laboratory, Department of Veterinary Population Medicine, University of Minnesota, 1333 Gortner Avenue Street, Paul, MN 55108, USA.
³ Department of Microbiology, University of Wisconsin-La Crosse, 1725 State Street, La Crosse, WI 54601, USA.

PMID: 25962537
DOI: 10.1016/j.jviromet.2015.04.025

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.

Keywords: Bluegill picornavirus; Eel picornavirus; Fathead minnow picornavirus; Multiplex RT-PCR; Reproducibility; Sensitivity; Specificity.

Publication types

Evaluation Study

MeSH terms

Animals
Fish Diseases / diagnosis*
Fish Diseases / virology
Great Lakes Region
Multiplex Polymerase Chain Reaction / methods*
Picornaviridae / genetics
Picornaviridae / isolation & purification*
Picornaviridae Infections / diagnosis
Picornaviridae Infections / veterinary*
Picornaviridae Infections / virology
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction / methods*
Sensitivity and Specificity
Veterinary Medicine / methods*