A specific and sensitive droplet digital polymerase chain reaction assay for the detection of tilapia lake virus in fish tissue and environmental samples

Abstract

Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify TiLV. The ddPCR assay could detect the virus at a lower threshold than the reverse transcription‐quantitative polymerase reaction (RT‐qPCR) method, and the sensitivity of the ddPCR assay was 10‐fold higher. The diagnostic sensitivity and specificity of the ddPCR assay were 100% and did not cross‐react with tilapia tissues infected with Tilapia parvovirus, Infectious spleen and kidney necrosis virus, Aeromonas hydrophila, Streptococcus agalactiae, S. iniae and Francisella noatunensis. The assay reproducibility was demonstrated by a high correlation coefficient of 0.998, and the inter‐assay coefficients of variability indicated that the ddPCR assay exhibited low variability within and between measurements. The detection limit of the TiLV ddPCR assay was 100 fg cDNA, which is equal to 3.3 copies of TiLV. Furthermore, the ddPCR assay could detect TiLV in mucus, water and infected tissue samples and the lowest copy number of TiLV detected in water samples by the ddPCR assay was 7.9 ± 0.99 copies/reaction The results of the clinical samples tested for TiLV revealed that the ddPCR assay had a relatively higher detection rate than the RT‐qPCR method. Overall, the ddPCR method offers a highly promising approach for the absolute quantification of TiLV in carrier fish and samples from the environment with low viral concentrations.

Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area

“…Therefore, the LOD reported by those authors would be between the LOD values we observed with RGNNV (0.1 cps/rctn) and SJNNV (6.8 cps/rctn). Similar LOD values have been reported by other authors for infectious spleen and kidney necrosis virus (3 cps/rctn; [16]), carp edema virus (2.2 cps/rctn; [17]), largemouth bass ranavirus (2.0 cps/rctn; [18]) or tilapia lake virus (3.3 cps/rtcn; [19]). A lower LOD, 0.07 cps/µL, has been reported for a method developed for tilapia parvovirus [20]; assuming a volume of 2 µL of template per ddRNA reaction mixture, that LOD would be equivalent to 0.14 cps/rctn, quite similar to what we obtained for the RG type with our protocol.…”