Development of Visual Loop-Mediated Isothermal Amplification Assays for Foodborne Hepatitis A Virus.

(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(0) 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.