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.
Jaroenram Wansadaj, Chatnuntawech Itthi, Kampeera Jantana, Pengpanich Sukanya, Leaungwutiwong Pornsawan, Tondee Benyatip, Sirithammajak Sarawut, Suvannakad Rapheephat, Khumwan Pakapreud, Dangtip Sirintip, Arunrut Narong, Bantuchai Sirasate, Nguitragool Wang, Wongwaroran Suchawit, Khanchaitit Paisan, Sattabongkot Jetsumon, Teerapittayanon Surat, Kiatpathomchai Wansika
Colorimetric RT-LAMP, LAMP-DETR, Machine learning, SARS-CoV-2