A droplet digital PCR method(ddPCR)for sensitive detection and quantification of Carassius auratus herpesvirus(CaHV)  

作　　者：Yun Zhao Dan Xu Fei Ke Yan Zhou Mingyou Li Lang Gui 

机构地区：[1]Key Laboratory of Freshwater Aquatic Genetic Resources Ministry of Agriculture and Rural Affairs,Shanghai Ocean University,Shanghai,China [2]Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai,China [3]International Research Center for Marine Biosciences,Ministry of Science and Technology,Shanghai Ocean University,Shanghai,China [4]State Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan,China

出　　处：《Water Biology and Security》2024年第2期55-61,共7页水生生物与安全（英文）

基　　金：financially supported by the Science and Technology Commission of Shanghai Municipality(21ZR1427200).

摘　　要：Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,is an urgent requirement for the prevention of CaHV transmission.In the present study,a droplet digital PCR(ddPCR)method based on the tumor necrosis factor receptor(TNFR)gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses.Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection,and positive amplification was detected on the first day by ddPCR(0.54 copies/μL),whereas the presence of CaHV was not detected by routine PCR until Day 6.Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment.The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR(qPCR),respectively.The results showed that the positive detection rate of ddPCR(100%)was higher than that of qPCR(40%).The detection limit of the ddPCR was found to be 0.52 copies/μL,which was much lower than the 50.12 copies/μL determined by qPCR.Overall,ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.

关 键 词：Carassius auratus herpesvirus(CaHV) Tumor necrosis factor receptor(TNFR) Droplet digital PCR(ddPCR) Real-time PCR(qPCR) Diagnosis method 

分 类 号：S94[农业科学—水产养殖]