Accurate Detection and Quantification of the Fish Viral Hemorrhagic Septicemia virus (VHSv) with a Two-Color Fluorometric Real-Time PCR Assay

作者：Pierce Lindsey R; Willey James C; Palsule Vrushalee V; Yeo Jiyoun; Shepherd Brian S; Crawford Erin L; Stepien Carol A^*

来源：PLos One, 2013, 8(8): e71851.

DOI：10.1371/journal.pone.0071851

Viral Hemorrhagic Septicemia virus (VHSv) is one of the world%26apos;s most serious fish pathogens, infecting %26gt;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.

• 出版日期2013-8-20