Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.

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

Mean log numbers of VHSv molecules/10⁶<i>actb1</i> molecules from our new 2-color fluorometric assay versus the prior Agilent capillary electrophoresis approach.

<p>Results indicate a linear relationship between the two tests (<i>R</i>² = 0.91, df = 1, 38, <i>F = </i>396.40, <i>p</i><0.001) and do not significantly differ (<i>t = </i>1.42, df = 78, NS). Arrow = Estimated threshold concentration of VHSv for fish with clinical signs of infection using our new assay, from a <i>χ</i>² test of nine symptomatic fish (1×10³ VHSv molecules/10⁶<i>actb1</i> molecules = 3.6×10² VHSv molecules). Triangle = false negative range for SYBR® green qRT-PCR (1.0×10⁰–1.6×10² VHSv/10⁶<i>actb1</i> molecules = 0.6×10⁰–2.5×10² VHSv molecules). Square = false negative range for cell culture (1.0×10⁰–2.2×10³ VHSv/10⁶<i>actb1</i> molecules = 0.6×10⁰–6.1×10³ VHSv molecules.</p

Specificity of the 2-color fluorometric test.

<p>– = negative result (no amplification), + = positive result.</p><p>Isolates obtained from:</p>a<p>Western Fisheries Research Center, USGS, Seattle, WA, USA.</p>b<p>Cornell University College of Veterinary Medicine, Ithaca, NY, USA.</p>c<p>Finnish Food Safety Authority Evira, Finland.</p>d<p>Universidad de Santiago de Compostela, Spain.</p>e<p>Fisheries and Oceans Canada, Pacific Biological Station, BC, Canada.</p

Concentrations for the 2-color fluorometric VHSv assay.

<p>Dilution mixtures (A–H) used for the Internal Standards Mixture (ISM) <i>actb1</i>/VHSv are given in units of 10^x M.</p

Relative numbers of VHSv positives and negatives from our 2-color fluorometric and capillary electrophoresis StaRT-PCR assays, which indicates identical numbers of positives and negatives.

<p>Compared to these tests, for 43 fishes (25 positives, 18 negatives (including 5 laboratory controls)), (a) SYBR® green has 44% false negative error (<i>χ</i>² = 5.67, df = 1, <i>p</i> = 0.02), and cell culture has 56% error (<i>χ</i>² = 9.36, df = 1, <i>p</i> = 0.002). For 63 fish samples (39 positives, 24 negatives (including 11 laboratory controls)), (b) SYBR® green qRT-PCR has 33% false negative error (<i>χ</i>² = 5.37, df = 1, <i>p</i> = 0.02), whereas the 2-color fluorometric and capillary electrophoresis tests show zero false negatives.</p

Relationship between the numbers of observed versus expected molecules when NT:IS concentrations are 1∶1.

<p>Results are based on dilutions of the Native Template (NT) and Internal Standard (IS) of 6×10⁶, 6×10⁵, 6×10⁴, 6×10³, 6×10², 60, 6, and 0.6 molecules for VHSv and <i>actb1</i>. The 2-color fluorometric assay yields a linear relationship for (a) VHSv over seven orders of magnitude (from 6×10⁶ to 6×10⁰ VHSv molecules) with a slope of 1.00 (<i>R</i>² = 0.99, <i>F = </i>9404.00, df = 1, 5, <i>p</i><0.001), and mean CV of 9%. A linear trend also is obtained for (b) <i>actb1</i> (<i>R</i>² = 0.99, <i>F = </i>1347.00, df = 1, 5, <i>p</i><0.001). Slope = 1.04, mean CV = 10%. Error bars = standard error of triplicate runs.</p

Sequences and PCR parameters for our 2-color fluorometric VHSv assay.

<p>Primers, probes, internal standards (IS), and synthetic templates are specified. F = forward primer, R = reverse primer, NT = native template. <i>Italics</i> = modified nucleotides in NT probe.</p

Relationship between the number of observed and expected molecules for NT:IS concentrations of 1∶1–1∶20.

<p>The concentration of Native Template (NT) is held constant and the Internal Standard (IS) varied for dilutions of: 1∶1 (6×10⁴ molecules), 1∶2, 1∶3, 1∶4, 1∶5, 1∶6, 1∶7, 1∶8, 1∶9, 1∶10, 1∶12, 1∶14, 1∶16, 1∶18, and 1∶20 (3×10³molecules). The 2-color fluorometric assay yields a linear relationship for (a) VHSv (<i>R</i>² = 0.99, <i>F = </i>1514.00, df = 1, 13, <i>p</i><0.001) with a mean CV of 5% for dilutions 1∶1–1∶10 and 7% for concentrations down to 1∶20, and for (b) <i>actb1</i> (<i>R</i>² = 0.99, <i>F = </i>1283.00, df = 1, 13, <i>p</i><0.001), CV = 5% and 7%. The same linear pattern is observed when the IS was held constant and NT varied for (c) VHSv (<i>R</i>² = 0.99, <i>F = </i>5124.00, df = 1, 13, <i>p</i><0.001), CV = 5% for 1∶1–1∶10 and 7% for dilutions down to 1∶20, and (d) <i>actb1</i> (<i>R</i>² = 0.99, <i>F = </i>2434.00, df = 1, 13, <i>p</i><0.001), CV = 3% and 6%. Error bars = standard error of results for triplicate runs. Dotted line = partition of dilutions from 1∶1–1∶10 (right) and 1∶12–1∶20 (left).</p

Real-time PCR amplification plots for various experiments.

<p>ABI 7500 real-time PCR results for (a) a true VHSv positive fish sample, (b) the relationship between the VHSv Native Template (NT) and Internal Standard (IS) with the NT held constant and the IS varied, (c) the relationship between VHSv NT and IS with the IS held constant and the NT varied, and (d) the relationship between VHSv NT:IS with concentrations held constant for dilutions of 1∶1–1∶20. Green = NT, red = IS, y = fluorescence of the reporter dye minus the baseline (Δ fluorescence), x = cycle threshold.</p

A New StaRT-PCR Approach to Detect and Quantify Fish Viral Hemorrhagic Septicemia Virus (VHSv): Enhanced Quality Control with Internal Standards

Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world\u27s most important finfish diseases, killing \u3e80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR® green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR®green real time qRT-PCR tests, and 47–70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0 × 100–1.2 × 105 VHSv/106actb1molecules in wild caught fishes and 1.0 × 100–8.4 × 105 molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean = 1.9 × 104) than those without (1.1 × 103) (p \u3c 0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv