A TaqMan RT-qPCR assay for tilapia lake virus (TiLV) detection in tilapia

Tilapia lake virus disease (TiLVD) caused by tilapia lake virus is a highly contagious disease affecting tilapia and its hybrid species. Thus far, the virus has been identified in >10 countries across 3 continents. For this reason, reliable and rapid diagnostic assays are urgently required. Here, we describe the development and validation of a TaqMan probe-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay using TiLV-93F/TiLV-93R primers and a TiLV93Probe targeting segment 3 of TiLV. The standard curve method was used to determine a PCR amplification efficiency of 92.9% over a wide linear range of 2.7 × 104 to 2.7 × 1010 TiLV copies. The intra-assay and inter-assay coefficients of variation are in the ranges of 0.54-2.18% and 0.59-3.88%, respectively. The TaqMan assay detected TiLV at 2.45 × 105 to 2.45 × 108 copy numbers in liver samples from 17 Nile and red hybrid tilapia samples from 2 countries and showing clinical signs of TiLVD. No fluorescence and thus no TiLV detection was found in liver samples from 11 healthy Nile and red hybrid tilapia. The specificity of the assay was further demonstrated by its inability to detect specimens known to have been infected with common fish pathogens, such as, Iridovirus, Streptococcus agalactiae, Francisella spp., Flavobacterium spp., and Aeromonas hydrophila. This newly developed TaqMan RT-qPCR assay can be used as an essential tool for TiLV diagnostics in disease surveillance and control programs, as well as in TiLV basic research projects.