Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand.
J Fish Dis. 2023 Sep;46(9):957-966. doi: 10.1111/jfd.13816. Epub 2023 Jun 9.
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.
罗非鱼湖病毒（TiLV）导致许多国家养殖和野生罗非鱼死亡率升高。我们开发了一种高特异性和高灵敏度的数字式 PCR（ddPCR）检测方法，用于检测和定量 TiLV。ddPCR 检测方法比反转录定量 PCR（RT-qPCR）方法的检测下限更低，灵敏度高 10 倍。ddPCR 检测方法的诊断灵敏度和特异性均为 100%，与感染了小RNA 病毒、传染性脾肾坏死病毒、嗜水气单胞菌、无乳链球菌、杀鲑气单胞菌和鳗弧菌的罗非鱼组织无交叉反应。ddPCR 检测方法的重现性通过高相关系数 0.998 来证明，组内和组间变异系数表明该 ddPCR 检测方法的变异度低。TiLV ddPCR 检测方法的检测限为 100 fg cDNA，相当于 3.3 拷贝 TiLV。此外，ddPCR 检测方法可检测粘液、水和感染组织样本中的 TiLV，ddPCR 检测方法在水样中检测到的 TiLV 最低拷贝数为 7.9±0.99 拷贝/反应。用 TiLV 检测临床样本的结果表明，ddPCR 检测方法的检测率相对高于 RT-qPCR 方法。总的来说，ddPCR 方法为定量检测低病毒浓度下的携带鱼和环境样本中的 TiLV 提供了一种很有前景的方法。
