Fisheries and Aquatic Sciences

The Korean Society of Fisheries and Aquatic Science (한국수산과학회)
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Evaluation of a novel TaqMan probe-based real-time polymerase chain reaction (PCR) assay for detection and quantitation of red sea bream iridovirus

  • Kim, Guk Hyun (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Kim, Min Jae (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Choi, Hee Ju (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Koo, Min Ji (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Kim, Min Jeong (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Min, Joon Gyu (Department of Aquatic Life Medicine, Pukyong National University) ;
  • Kim, Kwang Il (Department of Aquatic Life Medicine, Pukyong National University)
  • Received : 2021.05.07
  • Accepted : 2021.07.21
  • Published : 2021.11.30
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Abstract

The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD95%) was determined to be 5.3 viral genome copies/µL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.

Keywords

Acknowledgement

This work was supported by a Research Grant of Pukyong National University (2020).

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