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Development of a droplet digital PCR assay for the sensitive detection of iridovirus in Andrias davidianus

文献类型： 外文期刊

作者： Meng, Yan 1 ; Jiang, Nan 1 ; Xie, Yixing 2 ; Wei, Ying 2 ; Wang, Cheng 2 ; Tian, Mingzhu 2 ; Xue, Mingyang 1 ; Xu, Chen 1 ; Li, Yiqun 1 ; Liu, Wenzhi 1 ; Fan, Yuding 1 ; Zhou, Yong 1 ;

作者机构： 1.Chinese Acad Fishery Sci, Yangtze River Fisheries Res Inst, Wuhan, Peoples R China

2.Zhangjiajie Giant salamander Natl Nat Reserve Affa, Zhangjiajie, Peoples R China

关键词： Andrias davidianus; detection; droplet digital PCR; iridovirus

期刊名称：JOURNAL OF FISH DISEASES （ 影响因子：2.5； 五年影响因子：2.6 ）

ISSN： 0140-7775

年卷期： 2023 年

页码：

收录情况： SCI

摘要： Chinese giant salamander iridovirus (GSIV) is the first known and causative viral pathogen in Andrias davidianus. Developing a sensitive, accurate and specific assay to detect GSIV in samples is essential to prevent the further spread of the pathogen. In this study, we established a droplet digital PCR (ddPCR) assay that targeted the mcp gene of GSIV, enabling rapid and quantitative detection of the virus. We determined that the optimal annealing temperature, primer concentration and probe concentration were 57.1 degrees C, 50 nM and 500 nM, respectively. We analysed the specificity and sensitivity of the ddPCR assay and found that five common aquatic animal viruses, including Cyprinid herpesvirus 2 (CyHV-2), infectious spleen and kidney necrosis virus (ISKNV), Koi herpesvirus (KHV) and Carp Edema Virus (CEV) displayed negative results based on this GSIV ddPCR assay. The assay can detect GSIV with the lowest detection limit of 3.7 copies per reaction. To evaluate the sensitivity and accuracy of the ddPCR assay, we tested different infected tissue samples with both the ddPCR and TaqMan real-time PCR assays. Our results showed that the ddPCR assay detected GSIV in all samples with 100% positivity, while the TaqMan real-time PCR assay detected GSIV in only 82.1% of samples. The established ddPCR method provided several advantages in detecting GISV, including high sensitivity, high precision and absolute quantification, making it a powerful tool for detection of possible and potential GSIV infection, even in samples with low viral load.

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