Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols

Janet V. Warg, Travis Clement, Emily R. Cornwell, Angela Cruz, Rodman G. Getchell, Cem Giray, Andrew E. Goodwin, Geoffrey H. Groocock, Mohamed Faisal, Robert Kim, Gwenn E. Merry, Nicholas B D Phelps, Monica M. Reising, Isaac Standish, Yan Zhang, Kathy L. Kurth

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

Eight laboratories worked collectively to evaluate 4 real-time RT-PCR (rRT-PCR) protocols targeting viral hemorrhagic septicemia virus (VHSV) being considered for deployment to a USA laboratory testing network. The protocols utilized previously published primers and probe sets developed for detection and surveillance of VHSV. All participating laboratories received and followed a standard operating protocol for extraction and for each of the rRT-PCR assays. Performance measures specifically evaluated included limit of detection (defined as the smallest amount of analyte in which 95% of the samples are classified as positive), analytical specificity, assay efficiency across genotype representatives, within- and between-plate variation within a laboratory, and variation between laboratories using the same platform, between platforms, and between software versions. This evaluation clearly demonstrated that the TaqMan®-based assay developed by Jonstrup et al. (2013; J Fish Dis 36:9-23) produced the most consistent analytical performance characteristics for detecting all genotypes of VHSV across the 8 participating laboratories.

Original languageEnglish (US)
Pages (from-to)1-13
Number of pages13
JournalDiseases of Aquatic Organisms
Volume111
Issue number1
DOIs
StatePublished - Aug 21 2014
Externally publishedYes

Keywords

  • Analytical sensitivity
  • Analytical specificity
  • Real-time RT-PCR
  • Surveillance
  • Validation
  • VHSV

ASJC Scopus subject areas

  • Aquatic Science
  • Ecology, Evolution, Behavior and Systematics
  • Medicine(all)

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    Warg, J. V., Clement, T., Cornwell, E. R., Cruz, A., Getchell, R. G., Giray, C., Goodwin, A. E., Groocock, G. H., Faisal, M., Kim, R., Merry, G. E., Phelps, N. B. D., Reising, M. M., Standish, I., Zhang, Y., & Kurth, K. L. (2014). Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols. Diseases of Aquatic Organisms, 111(1), 1-13. https://doi.org/10.3354/dao02753