This study investigates the performance of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the colorimetric detection of SARS-CoV-2 using fluorometric dye, namely, calcein. The detection limit (LoD) with the N-ID1 primer set resulted in superior performance, corresponding to ~ 2 copies/reaction or ~ 0.1 copies/μL of the RNA sample. The color development can be observed by the naked eye, using an ultraviolet (UV) transilluminator or a hand-UV light without the requirement of expensive devices. The average time-to-reaction (TTR) value was 26.2 min in high-copy number samples, while it was about 50 min in rRT-PCR. A mobile application was proposed to quantify the positive and negative results based on the three-color spaces (RGB, Lab, and HSB). Compared to rRT-PCR (n = 67), this assay allows fast and sensitive visual detection of SARS-CoV-2, with high sensitivity (90.9%), selectivity (100%), and accuracy (94.03%). Besides, the assay was sensitive regardless of variants. Since this assay uses a fluorescent dye for visual observation, it can be easily adapted in RT-LAMP assays with high sensitivity. Thus, it can be utilized in low-source centers and field testing such as conferences, sports meetings, refugee camps, companies, and schools.

本研究探讨了逆转录环介导等温扩增 (RT-LAMP) 测定法使用荧光染料（即钙黄绿素）对 SARS-CoV-2 进行比色检测的性能。N-ID1 引物组的检测限 (LoD) 具有卓越的性能，相当于约 2 个拷贝/反应或约 0.1 个拷贝/μL 的 RNA 样品。可以使用紫外线 (UV) 透照器或手动紫外线灯通过肉眼观察颜色变化，无需昂贵的设备。高拷贝数样本中的平均反应时间 (TTR) 值为 26.2 分钟，而 rRT-PCR 中的平均反应时间 (TTR) 约为 50 分钟。提出了一个移动应用程序来量化基于三色空间（RGB、Lab 和 HSB）的正面和负面结果。与 rRT-PCR ( n = 67) 相比，该检测可以快速、灵敏地目视检测 SARS-CoV-2，具有高灵敏度 (90.9%)、选择性 (100%) 和准确性 (94.03%)。此外，无论变异如何，该测定都是敏感的。由于该测定使用荧光染料进行目视观察，因此可以轻松适应高灵敏度的 RT-LAMP 测定。因此，它可以用于会议、运动会、难民营、公司和学校等低资源中心和现场测试。