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 ampli...
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2025-03-01
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| author | Tongcan An Mengyuan Song Xiang Li Yingjie Pan Yong Zhao Haiquan Liu |
| author_facet | Tongcan An Mengyuan Song Xiang Li Yingjie Pan Yong Zhao Haiquan Liu |
| author_sort | Tongcan An |
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| description | (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<sup>0</sup> 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. |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-03-01 |
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| series | Foods |
| spelling | doaj-art-cbf126bd33f6444b853a7457ac0ee7ca2025-08-20T03:43:16ZengMDPI AGFoods2304-81582025-03-0114693410.3390/foods14060934Development of Visual Loop-Mediated Isothermal Amplification Assays for Foodborne Hepatitis A VirusTongcan An0Mengyuan Song1Xiang Li2Yingjie Pan3Yong Zhao4Haiquan Liu5College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, ChinaCollege of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, ChinaTechnical Center for Animal, Plant and Food Inspection and Quarantine of Shanghai Customs, Shanghai 201315, ChinaCollege of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, ChinaCollege of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, ChinaCollege of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China(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<sup>0</sup> 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.https://www.mdpi.com/2304-8158/14/6/934hepatitis A virusvisualization detectionlateral flow dipstickcolorimetric method |
| spellingShingle | Tongcan An Mengyuan Song Xiang Li Yingjie Pan Yong Zhao Haiquan Liu Development of Visual Loop-Mediated Isothermal Amplification Assays for Foodborne Hepatitis A Virus Foods hepatitis A virus visualization detection lateral flow dipstick colorimetric method |
| title | Development of Visual Loop-Mediated Isothermal Amplification Assays for Foodborne Hepatitis A Virus |
| title_full | Development of Visual Loop-Mediated Isothermal Amplification Assays for Foodborne Hepatitis A Virus |
| title_fullStr | Development of Visual Loop-Mediated Isothermal Amplification Assays for Foodborne Hepatitis A Virus |
| title_full_unstemmed | Development of Visual Loop-Mediated Isothermal Amplification Assays for Foodborne Hepatitis A Virus |
| title_short | Development of Visual Loop-Mediated Isothermal Amplification Assays for Foodborne Hepatitis A Virus |
| title_sort | development of visual loop mediated isothermal amplification assays for foodborne hepatitis a virus |
| topic | hepatitis A virus visualization detection lateral flow dipstick colorimetric method |
| url | https://www.mdpi.com/2304-8158/14/6/934 |
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