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Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus

文献类型： 外文期刊

作者： Jiang, Nan 1 ; Shen, Jinyu 2 ; Zhou, Yong 1 ; Liu, Wenzhi 1 ; Meng, Yan 1 ; Li, Yiqun 1 ; Xue, Mingyang 1 ; Xu, Chen 1 ; Fan, Yuding 1 ;

作者机构： 1.Chinese Acad Fishery Sci, Yangtze River Fisheries Res Inst, Fish Dis Div, Wuhan 430223, Hubei, Peoples R China

2.Zhejiang Inst Freshwater Fisheries, Agr Minist, Key Lab Healthy Freshwater Aquaculture, Key Lab Fish Hlth & Nutr Zhejiang Prov, Huzhou, Peoples R China

关键词： diagnosis method; droplet digital PCR; largemouth bass; largemouth bass ranavirus; major capsid protein

期刊名称：JOURNAL OF FISH DISEASES （ 影响因子：2.58； 五年影响因子：2.64 ）

ISSN： 0140-7775

年卷期：

页码：

收录情况： SCI

摘要： Largemouth bass ranavirus (LMBRaV), also known as largemouth bass virus (LMBV), is a high mortality pathogen in largemouth bass. A rapid, sensitive, specific and convenient diagnosis method is an urgent requirement for the prevention of virus transmission. In the present study, a droplet digital PCR (ddPCR) method based on the major capsid protein (mcp) gene was established to detect and quantify the virus genome copy number. Oligonucleotide primers were designed based on the LMBRaV mcp gene sequence. The specificity and sensitivity of ddPCR assay were analysed. The other aquatic virus including Chinese giant salamander iridovirus (GSIV), Cyprinid herpesvirus II (CyHV-2) and infectious spleen and kidney necrosis virus could not be detected by LMBRaV ddPCR assay. The detection limit of ddPCR assay was 2 +/- 0.37 copies/mu l DNA sample. And this ddPCR assay had great repeatability and reproducibility. In clinical diagnosis of 50 largemouth bass, 43 positive samples were detected by ddPCR, whereas only 34 positive samples were detected by quantitative PCR (qPCR). This LMBRaV detection assay provided a specific and sensitive method for the rapid diagnosis of LMBRaV infection in largemouth bass as well as quantification of the virus load.

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