Meng Yan, Jiang Nan, Xie Yixing, Wei Ying, Wang Cheng, Tian Mingzhu, Xue Mingyang, Xu Chen, Li Yiqun, Liu Wenzhi, Fan Yuding, Zhou Yong
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, China.
Zhangjiajie Giant salamander National Nature Reserve Affairs Center, Zhangjiajie, China.
J Fish Dis. 2023 Nov;46(11):1249-1256. doi: 10.1111/jfd.13844. Epub 2023 Aug 3.
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°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.
中国大鲵虹彩病毒（GSIV）是已知的大鲵体内的致病病毒病原体。开发一种灵敏、准确且特异的检测方法来检测样本中的GSIV对于防止该病原体的进一步传播至关重要。在本研究中，我们建立了一种针对GSIV的mcp基因的液滴数字PCR（ddPCR）检测方法，能够快速定量检测该病毒。我们确定最佳退火温度、引物浓度和探针浓度分别为57.1°C、50 nM和500 nM。我们分析了ddPCR检测方法的特异性和灵敏度，发现包括鲤疱疹病毒2型（CyHV-2）、传染性脾肾坏死病毒（ISKNV）、锦鲤疱疹病毒（KHV）和鲤水肿病毒（CEV）在内的五种常见水生动物病毒基于此GSIV ddPCR检测方法均显示阴性结果。该检测方法能够检测GSIV，最低检测限为每个反应3.7个拷贝。为了评估ddPCR检测方法的灵敏度和准确性，我们使用ddPCR和TaqMan实时PCR检测方法对不同的感染组织样本进行了检测。我们的结果表明，ddPCR检测方法在所有样本中均检测到GSIV，阳性率为100%，而TaqMan实时PCR检测方法仅在82.1%的样本中检测到GSIV。所建立的ddPCR方法在检测GISV方面具有多项优势，包括高灵敏度、高精度和绝对定量，使其成为检测可能和潜在GSIV感染的有力工具，即使在病毒载量较低的样本中也是如此。
