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Diseases of Aquatic Organisms

    DAO prepress abstract   -  DOI: https://doi.org/10.3354/dao03700

    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 Gustafson, Thomas Waltzek*

    *Corresponding author:

    ABSTRACT: Tilapia lake virus disease (TiLVD) is an emerged 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 polymerase chain reaction (RT-qPCR) assay for TiLV, targeting a conserved region within the 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-assay and inter-assay variability ranged from 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 determing 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.