A Rapid RT-LAMP Assay for SARS-CoV-2 with Colorimetric Detection Assisted by a Mobile Application

Abstract

Loop-mediated amplification has been promoted for SARS-CoV-2 screening, however, antigen tests are preferred in low-income countries and remote zones. Poor training in molecular biology, plus the need for RNA purification or reading instruments to overcome issues of sensitivity in colorimetric detection, are some of the reasons limiting the use of this technique. In this study, nasopharyngeal swabs, aspirates and saliva were amplified in an in-house LAMP assay and subject to colorimetric detection, achieved by the naked eye and by image analysis with a mobile application. Accuracy of detection by the naked eye ranged from 61–74% but improved to 75–86% when using the application. Sensitivity of the digital approach was 81% and specificity 83%, with poor positive predictive value, and acceptable negative predictive value. Additionally to the reported effect of some transport media’s pH, the presence of mucus and warming up of reagents while setting up the reaction critically affected performance. Accuracy per type of sample was 55, 70 and 80%, for swabs, aspirates and saliva, respectively, suggesting potential to improve the test in saliva. This assay, carried out in a closed tube, reduces contamination, has few pipetting steps and requires minimal equipment. Strategies to improve performance and implications of the use this sort of colorimetric LAMP for massive testing are discussed.

Clinical Validation of a Colorimetric Loop-Mediated Isothermal Amplification Using a Portable Device for the Rapid Detection of SARS-CoV-2

“…In addition, for each color capture (every 10 s) a standardized white light is emitted, guaranteeing that the same color pattern was captured. This is a clear advantage over smartphone capture, since the lightning exposure and equipment brand has a strong influence on the data acquisition [ 23 ]. Another advantage of our device is that it captures the data automatically.…”

“…We observed that if these steps weren’t followed, the RT-LAMP reaction color rapidly turned from pink to orange, indicating acidification of the solution due to the incorporation of atmospheric CO 2 . Another study that performed some of these procedures also saw an improvement in the stability of the color reaction [ 23 ].…”

Comparative Clinical Evaluation of a Novel FluA/FluB/SARS-CoV-2 Multiplex LAMP and Commercial FluA/FluB/SARS-CoV-2/RSV RT-qPCR Assays

“…The LAMP assay has several advantages over other nucleic acid amplification methods, such as being simple, sensitive, and rapid [ 12 ]. In addition, LAMP assays can be performed using a variety of detection methods, including colorimetric, lateral flow, fluorescence, and fluorescent probe detection [ 13 , 14 , 15 , 16 ]. Among these methods, fluorescent probe detection is particularly useful for the real-time monitoring of the LAMP reaction.…”

Development and Validation of Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) as a Simple and Rapid Diagnostic Tool for SARS-CoV-2 Detection

“…The overall detection accuracy was enhanced when using the mobile application (75–86%) compared to the naked eye (61–74%). In contrast, the detection accuracy was 55, 70, and 80% for the nasopharyngeal swabs, aspirates, and saliva, respectively [ 22 ]. The abovementioned studies illustrated the possibility of using RT-LAMP with different sampling locations than the naso-oropharyngeal swabs.…”