Lou Haoyu, Li Xuan, Wang Guohao, Zhang Kaisong, Wang Kejun, Tang Qiongying, Yang Guoliang, Jia Peng, Xiong Jinbo, Huang Jie, Dong Xuan
State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Qingdao, China.
College of Life Sciences, Huzhou University, Huzhou, China.
Appl Environ Microbiol. 2025 Aug 12:e0059125. doi: 10.1128/aem.00591-25.
The World Organisation for Animal Health (WOAH) has assessed crustacean diseases, such as infections with white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), decapod iridescent virus 1 (DIV1), and acute hepatopancreatic necrosis disease (AHPND), as listed diseases, and infection with (EHP) as an emerging disease, all of which significantly threaten the shrimp industry. This study developed a quintuplex EvaGreen-based melting curve real-time PCR method for the simultaneous detection of WSSV, IHHNV, DIV1, AHPND-causing (), and EHP. In the specific assay, only the target pathogen demonstrated efficient and detectable amplification, thereby indicating that the method exhibits high specificity. Regarding sensitivity testing, the five pathogens were detected at a concentration of 1.0 × 10 copies/μL. Each concentration gradient was evaluated in triplicate, and the coefficient of variation for each gradient remained below 3.38%, thereby affirming that the method demonstrates highly repeatability. We tested the diagnostic sensitivity (DSe) and the diagnostic specificity (DSp) of our method using a total of 800 clinical samples which were gathered from the shrimp farming regions in China. The newly established method in this study demonstrated a DSe above 89.74% for the five pathogens and a DSp of 100%. The quintuplex EvaGreen real-time PCR method developed here offers an accurate and efficient approach for EHP, WSSV, , IHHNV, and DIV1.IMPORTANCECrustacean diseases, such as infections with WSSV, IHHNV, DIV1, , and EHP, pose a significant threat to the global shrimp industry, leading to substantial economic losses. Rapid, accurate, and simultaneous detection of these pathogens is crucial for effective disease management and biosecurity in shrimp farming. In this study, we developed a quintuplex EvaGreen-based melting curve real-time PCR method that enables the simultaneous detection of these five major shrimp pathogens with exceptional specificity, sensitivity, and repeatability. By evaluating 800 clinical samples, our method demonstrated high diagnostic sensitivity and specificity, making it a valuable tool for early pathogen detection and disease control. This novel approach can help mitigate disease outbreaks, improve shrimp farm productivity, and support the sustainable development of the aquaculture industry.
世界动物卫生组织（WOAH）已将对虾疾病评估为法定报告疾病，如感染白斑综合征病毒（WSSV）、传染性皮下和造血组织坏死病毒（IHHNV）、十足目虹彩病毒1型（DIV1）以及急性肝胰腺坏死病（AHPND），并将感染肝肠胞虫（EHP）评估为一种新出现疾病，所有这些疾病都对虾产业构成重大威胁。本研究开发了一种基于五重EvaGreen熔解曲线的实时荧光定量PCR方法，用于同时检测WSSV、IHHNV、DIV1、引起急性肝胰腺坏死病的副溶血弧菌（VPAHPND）和EHP。在特异性检测中，只有目标病原体表现出高效且可检测到的扩增，这表明该方法具有高度特异性。在灵敏度测试中，这五种病原体在浓度为1.0×10拷贝/μL时均可被检测到。每个浓度梯度均进行了三次重复评估，每个梯度的变异系数均低于3.38%，从而证实该方法具有高度重复性。我们使用从中国对虾养殖区采集的总共800份临床样本，对我们方法的诊断敏感性（DSe）和诊断特异性（DSp）进行了测试。本研究新建立的方法对这五种病原体的DSe高于89.74%，DSp为100%。这里开发的五重EvaGreen实时荧光定量PCR方法为检测EHP、WSSV、VPAHPND、IHHNV和DIV1提供了一种准确有效的方法。
对虾疾病，如感染WSSV、IHHNV、DIV1、VPAHPND和EHP，对全球对虾产业构成重大威胁，导致巨大经济损失。快速、准确且同时检测这些病原体对于对虾养殖中的有效疾病管理和生物安全至关重要。在本研究中，我们开发了一种基于五重EvaGreen熔解曲线的实时荧光定量PCR方法，该方法能够以卓越的特异性、灵敏度和重复性同时检测这五种主要的对虾病原体。通过对800份临床样本的评估，我们的方法显示出高诊断敏感性和特异性，使其成为早期病原体检测和疾病控制的宝贵工具。这种新方法有助于减轻疾病爆发，提高对虾养殖场的生产力，并支持水产养殖业的可持续发展。
