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 Toohey-Kurth

doi:10.3354/dao02753

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Diseases of Aquatic Organisms

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DAO 111:1-13 (2014) - DOI: https://doi.org/10.3354/dao02753

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

Janet V. Warg^1,*, Travis Clement², Emily R. Cornwell³, Angela Cruz¹, Rodman G. Getchell³, Cem Giray⁴, Andrew E. Goodwin⁵, Geoffrey H. Groocock³, Mohamed Faisal⁶, Robert Kim⁶, Gwenn E. Merry⁵, Nicholas B. D. Phelps⁷, Monica M. Reising⁸, Isaac Standish⁶, Yan Zhang⁹, Kathy Toohey-Kurth¹⁰

¹Diagnostic Virology Laboratory, National Veterinary Services Laboratories, VS, APHIS, USDA, Ames, Iowa 50010, USA
²Veterinary and Biomedical Sciences Department, Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, South Dakota 57007, USA
³Aquatic Animal Health Program, Department of Microbiology and Immunology, Cornell University, Ithaca, New York 14853, USA
⁴Kennebec River Biosciences, Richmond, Maine 04357, USA
⁵Aquaculture/Fisheries Center, University of Arkansas Pine Bluff, Pine Bluff, Arkansas 71601, USA
⁶Aquatic Animal Health Laboratory, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan 48824, USA
⁷Veterinary Diagnostic Laboratory, University of Minnesota, St. Paul, Minnesota 55108, USA
⁸Center for Veterinary Biologics, VS, APHIS, USDA, Ames, Iowa 50010, USA
⁹Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg, Ohio 43068, USA
¹⁰Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin, Madison, Wisconsin 53706, USA

*Corresponding author: janet.v.warg@aphis.usda.gov

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.

KEY WORDS: VHSV · Surveillance · Real-time RT-PCR · Analytical sensitivity · Analytical specificity · Validation

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Cite this article as: Warg JV, Clement T, Cornwell ER, Cruz A and others (2014) Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols. Dis Aquat Org 111:1-13. https://doi.org/10.3354/dao02753

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DAO 111:1-13 (2014) - DOI: https://doi.org/10.3354/dao02753

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

Janet V. Warg1,*, Travis Clement2, Emily R. Cornwell3, Angela Cruz1, Rodman G. Getchell3, Cem Giray4, Andrew E. Goodwin5, Geoffrey H. Groocock3, Mohamed Faisal6, Robert Kim6, Gwenn E. Merry5, Nicholas B. D. Phelps7, Monica M. Reising8, Isaac Standish6, Yan Zhang9, Kathy Toohey-Kurth10