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- Title
Accurate Detection and Quantification of the Fish Viral Hemorrhagic Septicemia virus (VHSv) with a Two-Color Fluorometric Real-Time PCR Assay.
- Authors
Pierce, Lindsey R.; Willey, James C.; Palsule, Vrushalee V.; Yeo, Jiyoun; Shepherd, Brian S.; Crawford, Erin L.; Stepien, Carol A.
- 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 (R2 = 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/106actb1 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.
- Subjects
EURASIA; NORTH America; HEMORRHAGIC septicemia; POLYMERASE chain reaction; MARINE biology; ESTUARINE biology; BIOTECHNOLOGY; FISHERY sciences; CELL culture; FISHES
- Publication
PLoS ONE, 2013, Vol 8, Issue 8, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0071851