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.

One-step colorimetric isothermal detection of COVID-19 with AI-assisted automated result analysis: A platform model for future emerging point-of-care RNA/DNA disease diagnosis.

Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the diagnosis of COVID-19 owing to their unmatched feasibility in low-resource settings. However, the vast majority of them are restricted to proprietary pH-sensitive dyes that limit downstream assay optimization or hinder efficient result interpretation. To address this problem, we developed a novel dual colorimetric RT-LAMP assay using in-house pH-dependent indicators to maximize the visual detection and assay simplicity, and further integrated it with the artificial intelligence (AI) operated tool (RT-LAMP-DETR) to enable a more precise and rapid result analysis in large scale testing. The dual assay leverages xylenol orange (XO) and a newly formulated lavender green (LG) dye for distinctive colorimetric readouts, which enhance the test accuracy when performed and analyzed simultaneously. Our RT-LAMP assay has a detection limit of 50 viral copies/reaction with the cycle threshold (Ct) value ≤ 39.7 ± 0.4 determined by the WHO-approved RT-qPCR assay. RT-LAMP-DETR exhibited a complete concordance with the results from naked-eye observation and RT-qPCR, achieving 100% sensitivity, specificity, and accuracy that altogether render it suitable for ultrasensitive point-of-care COVID-19 screening efforts. From the perspective of pandemic preparedness, our method offers a simpler, faster, and cheaper (∼$8/test) approach for COVID-19 testing and other emerging pathogens with respect to RT-qPCR.