title: Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.
abstract: Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R(2) = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/10(6) actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.
authors: Great Lakes Genetics/Genomics Laboratory, Lake Erie Center and Department of Environmental Sciences, The University of Toledo, Toledo, Ohio, United States of America.
authors: Lindsey R
authors: LR
authors: Pierce
authors: James C
authors: JC
authors: Willey
authors: Vrushalee V
authors: VV
authors: Palsule
authors: Jiyoun
authors: J
authors: Yeo
authors: Brian S
authors: BS
authors: Shepherd
authors: Erin L
authors: EL
authors: Crawford
authors: Carol A
authors: CA
authors: Stepien
mesh_terms: D000818:Animals; D029941:Fish Proteins / Q000235:genetics; D005470:Fluorometry; D005814:Genes, Viral; D031941:Hemorrhagic Septicemia, Viral / Q000175:diagnosis* / Q000821:virology; D057230:Limit of Detection; D025202:Molecular Diagnostic Techniques; D029141:Novirhabdovirus / Q000235:genetics*; D010473:Perciformes / Q000235:genetics / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards
chemical_list: D029941:Fish Proteins
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