Real-time triplex loop-mediated isothermal amplification (LAMP) using a turbidimeter for detection of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV)

Objective: The World Organization for Animal Health still regulates the infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp. The existing disease identification approach is time consuming, necessitates expensive equipment, and requires specialized expertise, thereby limiting the accessibility of shrimp disease screening on farms. Loop-mediated isothermal amplification (LAMP) is recognized for its ability to detect inhibitory substances with high sensitivity and specificity. Methods: We developed a real-time triplex LAMP assay that combines the simplicity of point-of-care testing with the accuracy of a turbidimeter. Using a set of three LAMP primers, our technology enables rapid DNA amplification in a single reaction within 45 min and with a low detection limit (10 copies/reaction). Result: We tested 192 shrimp samples from different sources and demonstrated the clinical utility of our method, achieving 100% specificity (95% confidence interval = 93.40-100.00%), 100% sensitivity (97.36-100.00%), and 100% accuracy (98.10-100.00%) in detecting IHHNV DNA, with a high Cohen's kappa value (1) compared to the standard quantitative polymerase chain reaction assay. Conclusion: The high technology readiness level of our method makes it a versatile platform for any real-time LAMP assay, and its low cost and simplicity make it well suited for fast deployment and use in shrimp farming.