Development and validation of a novel Taqman‐based real‐time RT‐PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus

Abstract

Viral haemorrhagic septicaemia (VHS) is a serious disease in several fish species. VHS is caused by the rhabdovirus viral haemorrhagic septicaemia virus (VHSV). To prevent spreading of the pathogen, it is important to use a fast, robust, sensitive and specific diagnostic tool to identify the infected fish. Traditional diagnosis based on isolation in cell culture followed by identification using, for example, ELISA is sensitive and specific but slow. By switching to RT-PCR for surveillance and diagnosis of VHS the time needed before a correct diagnosis can be given will be considerably shortened and the need for maintaining expensive cell culture facilities reduced. Here we present the validation, according to OIE guidelines, of a sensitive and specific Taqman-based real-time RT-PCR. The assay detects all isolates in a panel of 79 VHSV isolates covering all known genotypes and subtypes, with amplification efficiencies of approximately 100%. The analytical and diagnostic specificity of the real-time RT-PCR is close to 1, and the analytical and diagnostic sensitivity is comparable with traditional cell-based methods. In conclusion, the presented real-time RT-PCR assay has the necessary qualities to be used as a VHSV surveillance tool on par with cell culture assays.

Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. II. Diagnostic evaluation of two protocols

“…Laboratories followed standardized testing protocols supplied to all participants. Detailed information on each assay can be found in Jonstrup et al (2013) and Phelps et al (2012). , and the RNA sample.…”

“…In this manuscript, we report on a collaborative side-by-side evaluation of the TaqMan ® based assay developed by Jonstrup et al (2013; hereafter 'Jonstrup assay') and the 1-step format of Phelps et al (2012;'Phelps assay') that utilizes primers and probe developed by Garver et al (2011) in an effort to estimate the diagnostic sensitivity and specificity of each test procedure. The 2 assays were selected based on a comparison of analytical performance of 4 assays (Warg et al 2014, this volume).…”

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

“…Depending on the method used, virus yield from tissue samples may vary greatly, and there could be substantial variation in sensitivities with subsequent contamination (Whittington & Steiner 1993, Rimmer et al 2012. Likewise, diagnostic sensitivity of PCR-based assays may be affected by the choice of PCR reagents (Elliott et al 2013, Jonstrup et al 2013). 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.…”

“…as well as temporal changes in pathogen distribution in tissues and associated histopathological changes. The Jonstrup et al (2013) study described the use of samples from fish after an acute challenge experiment to validate a test for surveillance purposes. In that study, quantification of the viral pathogen by both the TUE and the RS was used to demonstrate the presence of a range of viral quantities, including samples close to the TUE's limit of detection.…”

“…For continuous outcomes, imprecision is most relevant for test values near the cutoff point and should be reported. The precision of continuous tests can be illustrated graphically across analyte concentration using a boxplot, concordance plot or Blandand-Altman (see, for example, Corbeil et al 2010, Garver et al 2011, and Jonstrup et al 2013). For binary outcome tests, a novel graphical approach using phylogenetic tree representation was developed to represent clustering (agreement) among test replicates (Caraguel et al 2009).…”