Development and validation of three duplex real-time PCR assays for simultaneous detection of major haemorrhagic disease-associated pathogens in cultured eels

IF 3.9 1区农林科学 Q1 FISHERIES

Aquaculture Pub Date : 2025-09-01 DOI:10.1016/j.aquaculture.2025.743148

Jing Xiong , Yi-xin Huang , Dong-li Li , Bei Huang , Ying Liang , Wen-shu Huang

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引用次数: 0

Abstract

Haemorrhagic disease causes significant losses in global eel aquaculture. To enable concurrent diagnosis of its diverse etiological agents, we developed three duplex TaqMan real-time PCR (qPCR) assays targeting: (1) Anguillid herpesvirus (AngHV) DNA polymerase gene (pol) and Eel circovirus (EeCV) capsid protein gene (cp); (2) Aeromonas cytotoxic enterotoxin (act) and heat-stable cytotonic enterotoxin (ast) genes; (3) Aeromonas heat-labile cytotoxic enterotoxin (alt) and hemolysin (hlyA) genes. Each assay was rigorously optimized and validated. Optimized performance was achieved using primer and probe concentrations of 0.2 μM and 0.1 μM, respectively, and a uniform annealing temperature of 60 °C for 20 s. All assays exhibited high specificity, efficient amplification (92.5–99.8 %), excellent linearity (R² ≥ 0.998), and high sensitivity, with limits of detection (LOD) of 6.2 copies/μL for pol and cp, and 3.6 copies/μL for the Aeromonas toxin genes (act, ast, alt and hlyA). Validation with fourteen Aeromonas strains demonstrated perfect concordance with singleplex qPCR. Applied to 54 clinical eel samples, mixed infections (56 %, 30/54), predominated over single infections (22 %, 12/54). Prevalence was EeCV (68.5 %), AngHV (55.6 %), and virulent Aeromonas spp. (44.4 %). Key co-infection patterns were AngHV + EeCV + Aeromonas spp. (35.2 %), AngHV + EeCV (11.1 %), and AngHV + Aeromonas spp. (9.3 %). Collectively, this study established the first duplex qPCR platform for simultaneous detection of AngHV, EeCV, and virulent Aeromonas spp. (via key toxin genes) associated with eel haemorrhagic disease, providing a powerful, high-throughput tool for rapid diagnosis and epidemiological surveillance.

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开发和验证三种双工实时PCR法同时检测养殖鳗鱼中主要出血性疾病相关病原体

出血性疾病对全球鳗鱼养殖业造成重大损失。为了能够同时诊断其多种病原，我们建立了三种双工TaqMan实时荧光定量PCR （qPCR）检测方法：(1)鳗鲡疱疹病毒（AngHV） DNA聚合酶基因（pol）和鳗鲡圆环病毒（EeCV）衣壳蛋白基因（cp）；(2)气单胞菌细胞毒性肠毒素（act）和热稳定型细胞滋补肠毒素（ast）基因；(3)气单胞菌热不稳定细胞毒性肠毒素（alt）和溶血素（hlyA）基因。每个实验都经过严格的优化和验证。引物和探针浓度分别为0.2 μM和0.1 μM，均匀退火温度为60°C，退火时间为20 s时，得到了最佳性能。结果表明，该方法特异性高，扩增效率高（92.5 ~ 99.8%），线性度高（R2≥0.998），对气单胞菌毒素基因（act、ast、alt和hlyA）的检出限为6.2 copies/μL，对pol和cp的检出限为3.6 copies/μL。对14株气单胞菌进行了qPCR验证，结果完全一致。54份临床鳗鱼样本中，混合感染占56%(30/54)，高于单一感染占22%（12/54）。流行率为EeCV（68.5%）、AngHV（55.6%）和强毒气单胞菌（44.4%）。主要共感染类型为AngHV + EeCV +气单胞菌（35.2%）、AngHV + EeCV（11.1%）和AngHV +气单胞菌（9.3%）。总之，本研究建立了首个同时检测与鳗鱼出血性疾病相关的AngHV、EeCV和毒力气单胞菌（通过关键毒素基因）的双工qPCR平台，为快速诊断和流行病学监测提供了强大的高通量工具。

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来源期刊

Aquaculture 农林科学-海洋与淡水生物学

CiteScore

8.60

自引率

17.80%

发文量

1246

审稿时长

56 days

期刊介绍： Aquaculture is an international journal for the exploration, improvement and management of all freshwater and marine food resources. It publishes novel and innovative research of world-wide interest on farming of aquatic organisms, which includes finfish, mollusks, crustaceans and aquatic plants for human consumption. Research on ornamentals is not a focus of the Journal. Aquaculture only publishes papers with a clear relevance to improving aquaculture practices or a potential application.