Rapid and user‐friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on‐site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual‐sample microfluidic chip integrated with a real‐time fluorogenic loop‐mediated isothermal amplification assay (dual‐sample on‐chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on‐chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 10⁰ to 10^–1 pg/μl for genomic DNA of tested bacteria and 10^–4 to 10^–5 pg/μl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time‐to‐positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on‐chip LAMP assay provides an effective dual‐sample and multiple pathogen analysis, and thus would be applicable to on‐site detection and routine monitoring of multiple pathogens in aquaculture.

中文翻译：

---

使用荧光环介导的等温扩增双样品微流控芯片同时检测多种细菌和病毒水生病原体

快速和用户友好的诊断测试对于疾病的早期诊断和立即发现是必要的，尤其是对于水产养殖中的病原微生物的现场筛选。在这项研究中，我们开发了一种双样品微流控芯片，该芯片与实时荧光环介导的等温扩增测定法（双样品片上LAMP）集成，可同时检测10种病原微生物，即嗜水气单胞菌，塔德氏菌，弧菌。哈维氏，溶藻弧菌，鳗弧菌，副溶血弧菌，创伤弧菌;，感染性皮下和造血细胞坏死病毒，感染性脾肾坏死病毒和白斑综合症病毒。该芯片上的LAMP提供了几乎自动化的协议，可以同时分析两个样品，并且测试达到了检测 细菌基因组DNA的检测限（LOD）从10 ⁰到10 ^–1 pg /μl，从10 ^–4到10到10 pg /μl。^–5 pg /μl用于测试病毒的重组质粒DNA，平均运行时间少于30分钟。正值时间的变异系数小于10％，反映了强大的可重复性。与传统的微生物学或临床方法相比，其临床敏感性和特异性分别为93.52％和85.53％。片上LAMP分析可提供有效的双样本和多种病原体分析，因此适用于水产养殖中多种病原体的现场检测和常规监测。