Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus

Abstract

Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) ≥93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.

Molecular epidemiology of viral haemorrhagic septicaemia virus (VHSV) in British Columbia, Canada, reveals transmission from wild to farmed fish

“…Initial virus identity for isolates prior to 1998 was determined using a DNA probe (Batts et al 1993), while isolations post 1998 were confirmed via VHSV-specific reverse transcriptase polymerase chain reaction (RT-PCR) as described below. All BC VHSV isolates listed herein were cultured with the exception of the Pa cific Hake isolate which was detected via quantitative RT-PCR (RT-qPCR) (Garver et al 2011) and RT-PCR.…”

Recommended reporting standards for test accuracy studies of infectious diseases of finfish, amphibians, molluscs and crustaceans: the STRADAS-aquatic checklist

“…For quality control purposes and to provide unambiguous definitions of positive and negative test results, both negative and positive controls should be included in each of the test runs. To show the needed information in this item, a description of the quality control samples used in a real-time RT-PCR validation study is presented in the example above (Garver et al 2011) and in Greer & Collins (2007). Authors should also specify whether or not internal and/or artificial controls are included in PCR assays to identify false-negative or falsepositive test results (Snow et al 2009, Purcell et al 2014, as well as the protocol for retesting such samples.…”

“…The challenge conditions, particularly the dose and strain of pathogen, should be reported to allow readers to evaluate how well the experimental model reflects natural exposure under field conditions. The examples by Greer & Collins (2007) and Garver et al (2011) include most of the necessary elements with the exception of Animal Ethics Committee approval of the experiment design. Research studies with amphibians and fish, but typically not molluscs and crustaceans, must be approved by the Animal Ethics Committee in most research institu-tions and should be reported as done in Purcell et al (2013), with the possible addition of approved protocol numbers (see Hyatt et al 2007).…”

“…Blinding avoids both potential bias in the interpretation of test results and overly optimistic estimates of diagnostic sensitivity and specificity or the area under the receiver-operating curve in diagnostic test evaluations. Procedures for blinding should be described as in the Garver et al (2011) example or as in Thébault et al (2005). If the joint results of the TUE and RS (positive−positive, positive−negative, negative−positive, and negative) are reviewed by testing laboratories and retesting of samples with discrepant (discordant or nonagreeing) results is done using a third test, estimates of sensitivity and specificity will change and may be inflated (Hadgu 1999).…”

“…kappa with 95% CI). For a review of methods for estimating and reporting agreement for continuous test outcomes, see for example, Barnhart et al (2007), Garver et al (2011), andOIE (2015c). Caraguel et al (2011) is a good example of reporting of more complex modelling methods to investigate the impact of sample or operational factors on test precision.…”