Development of a Novel Nested-RT-LAMP Assay for the Rapid and Accurate Coronavirus Disease-2019 Diagnosis.

Background and Aims: Coronavirus disease 2019 (COVID-19), an emerging life-threatening viral disease, has rapidly spread worldwide, exerting a detrimental impact on public health. We aimed to devise an innovative platform based on the loop-mediated isothermal amplification (LAMP) method, having priorities over real-time PCR (RT-PCR) in terms of sensitivity, specificity, and low running costs. Methods: To develop a novel assay, a new primer set plus four primer sets were designed targeting the N gene of the COVID-19 agent, resulting in the sensitivity reinforcement. The limit of detection (LOD) of the developed approach was determined and compared to those of the standard RT-LAMP and RT-PCR. Two hundred confirmed positive and negative samples initially tested by RT-PCR were recruited to assess the nested-RT-LAMP assay. Furthermore, for the one-step nested-RT-LAMP assay, positive samples were tested directly without the need for RNA extraction. Results: The LOD of nested-RT-LAMP, LAMP, and RT-PCR were 5, 15, and 15 copies/μL, respectively. The findings of the investigation illustrated 100% sensitivity and 98% specificity for both LAMP assays. Moreover, respectively, 94% and 97% sensitivity and specificity were determined regarding the one-step nested-RT-LAMP assay. Conclusion: We offered a novel approach with more sensitivity compared to RT-PCR and common RT-LAMP, not only being a simple, accurate, cost-effective alternative diagnostic tool for RT-PCR but also being able to detect asymptomatic or mildly symptomatic patients more accurately in 2 h by naked eyes.