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

Dorothea V. Megarani; Lowia Al-Hussinee; Kuttichantran Subramaniam; Preeyanan Sriwanayos; Kamonchai Imnoi; Bill Keleher; Pamela Nicholson; Win Surachetpong; Puntanat Tattiyapong; Paul Hick; Lori L. Gustafson; Thomas B. Waltzek

doi:10.3354/dao03700

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Diseases of Aquatic Organisms

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DAO 152:147-158 (2022) - DOI: https://doi.org/10.3354/dao03700

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

Dorothea V. Megarani^1,2, Lowia Al-Hussinee^1,2, Kuttichantran Subramaniam^1,2, Preeyanan Sriwanayos^1,2,8, Kamonchai Imnoi^1,2,8, Bill Keleher³, Pamela Nicholson⁴, Win Surachetpong⁵, Puntanat Tattiyapong⁵, Paul Hick⁶, Lori L. Gustafson⁷, Thomas B. Waltzek^1,2,9,*

¹Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610, USA
²Emerging Pathogens Institute, University of Florida, Gainesville, Florida 32610, USA
³Kennebec River Biosciences, Richmond, Maine 04357, USA
⁴Next Generation Sequencing Platform, University of Bern, Bern 3012, Switzerland
⁵Department of Veterinary Microbiology and Immunology, Kasetsart University, Bangkok 10900, Thailand
⁶Virology Laboratory, New South Wales Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, NSW 2568, Australia
⁷Animal and Plant Health Inspection Services, US Department of Agriculture, Fort Collins, Colorado 80526, USA
⁸Present address: Aquatic Animal Health Research and Development Division, Department of Fisheries, Bangkok 10900, Thailand
⁹Present address: Animal and Plant Health Inspection Services, US Department of Agriculture, Gainesville, Florida 32608, USA

*Corresponding author: tomwaltzek@gmail.com

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 93 proven-positive and 185 proven-negative samples yielded a diagnostic sensitivity of 96.8% 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.

KEY WORDS: Tilapia · Oreochromis spp. · Tilapia lake virus · Tilapia tilapinevirus · TaqMan · Quantitative PCR · Diagnostic accuracy

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Cite this article as: Megarani DV, Al-Hussinee L, Subramaniam K, Sriwanayos P and others (2022) Development of a TaqMan quantitative reverse transcription PCR assay to detect tilapia lake virus. Dis Aquat Org 152:147-158. https://doi.org/10.3354/dao03700

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DAO 152:147-158 (2022) - DOI: https://doi.org/10.3354/dao03700

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

Dorothea V. Megarani1,2, Lowia Al-Hussinee1,2, Kuttichantran Subramaniam1,2, Preeyanan Sriwanayos1,2,8, Kamonchai Imnoi1,2,8, Bill Keleher3, Pamela Nicholson4, Win Surachetpong5, Puntanat Tattiyapong5, Paul Hick6, Lori L. Gustafson7, Thomas B. Waltzek1,2,9,*

Dorothea V. Megarani^1,2, Lowia Al-Hussinee^1,2, Kuttichantran Subramaniam^1,2, Preeyanan Sriwanayos^1,2,8, Kamonchai Imnoi^1,2,8, Bill Keleher³, Pamela Nicholson⁴, Win Surachetpong⁵, Puntanat Tattiyapong⁵, Paul Hick⁶, Lori L. Gustafson⁷, Thomas B. Waltzek^1,2,9,*