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

• Published in: Talanta (Oxford) 2022-11, Vol.249, p.123375-123375, Article 123375
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: Artificial Intelligence; Colorimetric RT-LAMP; Colorimetry - methods; COVID-19 - diagnosis; COVID-19 Testing; DNA; Humans; LAMP-DETR; Machine learning; Nucleic Acid Amplification Techniques - methods; Point-of-Care Systems; RNA; RNA, Viral - genetics; SARS-CoV-2; SARS-CoV-2 - genetics; Sensitivity and Specificity
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2022.123375

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. [Display omitted] •We developed a novel ultrasensitive and specific dual RT-LAMP assay.•It targets the Nsp9 segment of SARS-CoV-2 ORF1ab and human 18 S rRNA gene.•Results can be analyzed with the naked-eye and an AI-operated automated model.•The assay can be exploited for decentralized high-throughput COVID-19 screening.