An Tongcan, Song Mengyuan, Li Xiang, Pan Yingjie, Zhao Yong, Liu Haiquan
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.
Technical Center for Animal, Plant and Food Inspection and Quarantine of Shanghai Customs, Shanghai 201315, China.
Foods. 2025 Mar 10;14(6):934. doi: 10.3390/foods14060934.
(1) Background: There are many cases of human disease caused by the hepatitis A virus contamination of aquatic products, so the development of the rapid detection of hepatitis A virus in aquatic products is crucial. (2) Methods: In this study, we developed three visual loop-mediated isothermal amplification methods for the rapid and intuitive detection of hepatitis A virus in aquatic products. New specific LAMP primers were designed for the HAV-specific VP1 protein shell. (1) HNB dye was added to the LAMP reaction system. After the reaction, the color of the reaction mixture changed from violet to sky blue, showing a positive result. (2) Cresol red dye was added to the LAMP reaction system, and a positive result was indicated by orange, while a negative result was indicated by purple. (3) By labeling FIP with biotin and LF with 6-FAM, the amplified product simultaneously contained biotin and 6-FAM, which bound to the anti-biotin antibody on the gold nanoparticles on the lateral flow dipstick (LFD). Subsequently, biotin was further combined with the anti-fam antibody on the T-line of the test strip to form a positive test result. (3) Results: The three visual LAMP methods were highly specific for HAV. The sensitivity of the visual assay was 2.59 × 10 copies/μL. The positive detection ratio for 155 bivalve shellfish samples was 8.39%, which was the same as that for RT-qPCR. The three visual LAMP methods established in our work have better sensitivity than the international gold standard, and their operation is simple and requires less time. (4) Conclusions: The results can be obtained by eye color comparison and lateral flow dipsticks. Without the use of large-scale instrumentation, the sensitivity is the same as that of RT-qPCR. The test strips are lightweight, small in size, and easy to carry; they are suitable for emergency detection, on-site monitoring, field sampling, or remote farms and other non-laboratory environments for rapid identification.
(1) 背景：水产品甲型肝炎病毒污染导致多起人类疾病病例，因此开发水产品中甲型肝炎病毒的快速检测方法至关重要。(2) 方法：在本研究中，我们开发了三种可视化环介导等温扩增方法，用于快速直观地检测水产品中的甲型肝炎病毒。针对甲型肝炎病毒特异性VP1蛋白衣壳设计了新的特异性LAMP引物。(1) 在LAMP反应体系中加入HNB染料。反应后，反应混合物颜色由紫色变为天蓝色，表明结果为阳性。(2) 在LAMP反应体系中加入甲酚红染料，橙色表示阳性结果，紫色表示阴性结果。(3) 通过用生物素标记FIP和用6-FAM标记LF，扩增产物同时含有生物素和6-FAM，它们与侧向流动试纸条（LFD）上金纳米颗粒上的抗生物素抗体结合。随后，生物素进一步与试纸条T线处的抗FAM抗体结合，形成阳性检测结果。(3) 结果：三种可视化LAMP方法对甲型肝炎病毒具有高度特异性。可视化检测的灵敏度为2.59×10拷贝/μL。155份双壳贝类样品的阳性检出率为8.39%，与RT-qPCR相同。我们建立的三种可视化LAMP方法比国际金标准具有更好的灵敏度，且操作简单、耗时少。(4) 结论：通过颜色目视比较和侧向流动试纸条即可获得结果。无需使用大型仪器，灵敏度与RT-qPCR相同。试纸条重量轻、体积小、便于携带；适用于应急检测、现场监测、野外采样或偏远养殖场等非实验室环境的快速鉴定。
