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

Autor: Nan Jiang, Jinyu Shen, Yong Zhou, Wenzhi Liu, Yan Meng, Yiqun Li, Mingyang Xue, Chen Xu, Yuding Fan
Rok vydání: 2022
Předmět:
Zdroj: Journal of Fish Diseases. 46:91-98
ISSN: 1365-2761
0140-7775
DOI: 10.1111/jfd.13721
Popis: 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/μl 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.
Databáze: OpenAIRE
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