Development of a TaqMan quantitative reverse transcription PCR assay to detect tilapia lake virus

Author:

Megarani DV12,Al-Hussinee L12,Subramaniam K12,Sriwanayos P123,Imnoi K123,Keleher B4,Nicholson P5,Surachetpong W6,Tattiyapong P6,Hick P7,Gustafson LL8,Waltzek TB129

Affiliation:

1. Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610, USA

2. Emerging Pathogens Institute, University of Florida, Gainesville, Florida 32610, USA

3. Aquatic Animal Health Research and Development Division, Department of Fisheries, Bangkok 10900, Thailand

4. Kennebec River Biosciences, Richmond, Maine 04357, USA

5. Next Generation Sequencing Platform, University of Bern, Bern 3012, Switzerland

6. Department of Veterinary Microbiology and Immunology, Kasetsart University, Bangkok 10900, Thailand

7. Virology Laboratory, New South Wales Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, NSW 2568, Australia

8. Animal and Plant Health Inspection Services, US Department of Agriculture, Fort Collins, Colorado 80526, USA

9. Animal and Plant Health Inspection Services, US Department of Agriculture, Gainesville, Florida 32608, USA

Abstract

Tilapia lake virus disease (TiLVD) is an emerging viral disease associated with high morbidity and mortality in cultured tilapia worldwide. In this study, we have developed and validated a TaqMan quantitative reverse transcription PCR (RT-qPCR) assay for TiLV, targeting a conserved region within segment 10 of the genome. The RT-qPCR assay was efficient (mean ± SD: 96.71 ± 3.20%), sensitive with a limit of detection of 10 RNA viral copies per reaction, and detected TiLV strains from different geographic regions including North America, South America, Africa, and Asia. The intra- and inter-assay variability ranged over 0.18-1.41% and 0.21-2.21%, respectively. The TaqMan RT-qPCR assay did not cross-react with other RNA viruses of fish, including an orthomyxovirus, a betanodavirus, a picornavirus, and a rhabdovirus. Analysis of 91 proven-positive and 185 proven-negative samples yielded a diagnostic sensitivity of 96.7% and a diagnostic specificity of 100%. The TaqMan RT-qPCR assay also detected TiLV RNA in infected Nile tilapia liver tissue extracts following an experimental challenge study, and it successfully detected TiLV RNA in SSN-1 (E-11 clone) cell cultures displaying cytopathic effects following their inoculation with TiLV-infected tissue homogenates. Thus, the validated TaqMan RT-qPCR assay should be useful for both research and diagnostic purposes. Additionally, the TiLV qPCR assay returns the clinically relevant viral load of a sample which can assist health professionals in determining the role of TiLV during disease investigations. This RT-qPCR assay could be integrated into surveillance programs aimed at mitigating the effects of TiLVD on global tilapia production.

Publisher

Inter-Research Science Center

Subject

Aquatic Science,Ecology, Evolution, Behavior and Systematics

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