Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of Infectious spleen and kidney necrosis virus

Samantha A Koda; Kuttichantran Subramaniam; Paul M Hick; Evelyn Hall; Thomas B Waltzek; Joy A Becker

doi:10.1371/journal.pone.0281292

Partial validation of a TaqMan quantitative polymerase chain reaction for the detection of the three genotypes of Infectious spleen and kidney necrosis virus

PLoS One. 2023 Feb 3;18(2):e0281292. doi: 10.1371/journal.pone.0281292. eCollection 2023.

Authors

Samantha A Koda^{1

2}, Kuttichantran Subramaniam^{1

2}, Paul M Hick³, Evelyn Hall³, Thomas B Waltzek^{1

2}, Joy A Becker⁴

Affiliations

¹ Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, United States of America.
² Emerging Pathogens Institute, University of Florida, Gainesville, Florida, United States of America.
³ Sydney School of Veterinary Science, The University of Sydney, Camden, New South Wales, Australia.
⁴ School of Life and Environmental Sciences, The University of Sydney, Camden, New South Wales, Australia.

Abstract

Megalocytiviruses (MCVs) are double-stranded DNA viruses known to infect important freshwater and marine fish species in the aquaculture, food, and ornamental fish industries worldwide. Infectious spleen and kidney necrosis virus (ISKNV) is the type species within the genus Megalocytivirus that causes red seabream iridoviral disease (RSIVD) which is a reportable disease to the World Animal Health Organization (WOAH). To better control the transboundary spread of this virus and support WOAH reporting requirements, we developed and partially validated a TaqMan real-time qPCR assay (ISKNV104R) to detect all three genotypes of ISKNV, including the two genotypes that cause RSIVD. Parameters averaged across 48 experiments used a 10-fold dilution series of linearized plasmid DNA (107-101 copies), carrying a fragment of the three-spot gourami iridovirus (TSGIV) hypothetical protein revealed that the assay was linear over 7 orders of magnitude (107-101), a mean efficiency of 99.97 ± 2.92%, a mean correlation coefficient of 1.000 ± 0.001, and a limit of detection (analytical sensitivity) of ≤10 copies of TSGIV DNA. The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was evaluated and compared to other published assays using a panel of 397 samples from 21 source populations with different prevalence of ISKNV infection (0-100%). The diagnostic sensitivity and specificity for the ISKNV104R qPCR assay was 91.99% (87.28-95.6; 95% CI) and 89.8% (83.53-94.84). The latent class analysis showed that the ISKNV104R qPCR assay had similar diagnostic sensitivities and specificities with overlapping confidence limits compared to a second TaqMan qPCR assay and a SYBR green assay. This newly developed TaqMan assay represents a partially validated qPCR assay for the detection of the three genotypes of the species ISKNV. The ISKNV104R qPCR assay once fully validated, will serve as an improved diagnostic tool that can be used for ISKNV surveillance efforts and diagnosis in subclinical fish to prevent further spread of MCVs throughout the aquaculture and ornamental fish industries.

Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
DNA Virus Infections* / diagnosis
DNA Virus Infections* / epidemiology
DNA Virus Infections* / veterinary
Fish Diseases* / epidemiology
Genotype
Iridoviridae* / genetics
Perciformes* / genetics
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Sea Bream* / genetics

Supplementary concepts

Infectious spleen and kidney necrosis virus

Grants and funding

The authors thank the Australian Government Department of Education and Training’s Enabling Growth and Innovation Project Fund for providing travel funding through their Australia-Americas PhD Research Internship Program (SAK). The collaboration between the University of Florida and University of Sydney would not have been possible without this. This study was partially funded by the Australian Government through the Fisheries Research and Development Corporation (FRDC) (Project No. 2014/001 and Project No. 2016-011) (JAB) and the University of Sydney (JAB, PMH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.