Phenol red as electrochemical indicator for highly sensitive quantification of SARS-CoV-2 by loop-mediated isothermal amplification detection.

The current COVID-19 pandemic has made patent the need for rapid and cost-effective diagnostic tests, crucial for future infectious outbreaks. Loop-mediated isothermal amplification (LAMP) is a promising and decentralized alternative to qPCR. In this work we have developed a sensitive, fast, and simple innovative methodology for quantification of SARS-CoV-2 RNA copies, combining reverse-transcription LAMP with electrochemical detection. This is based on the oxidation of phenol red (PR), a visual and electroactive LAMP indicator, which oxidation peak potential (E _p ) changes with the progress of the LAMP reaction. Using that E _p shift as analytical signal, a calibration curve was obtained for fragment N1 copies of SARS-CoV2 (which provided better results than N or S fragments), with a potential shift of 16.2 mV per order of magnitude, and a practical limit of detection of 21 copies·μL ^-1 . Moreover, the precision of E _p is excellent (RSD < 2%): 557 ± 5 mV for negative and 602 ± 7 mV for positive (2148 N fragment RNA copies·µL ^-1 · ^-1 ) LAMP controls. This methodology has been applied to the analysis of nasopharyngeal swab samples, resulting in total concordance with clinical RT-qPCR results. Advances towards fully decentralization have been achieved by designing and fabricating a small portable heater for isothermal procedures, obtaining comparable results to those from a commercial benchtop thermal cycler.
Competing Interests: Declaration of competing interest M.T.F.-A. declares she is one of the founders of Micrux Technologies, a company engaged in the development of miniaturized electrodes and electrochemical/microfluidic equipment and remains a consultant and shareholder. She declares it has not influenced the work reported in this paper. The rest of authors declare no competing financial interest.
(Copyright © 2023. Published by Elsevier B.V.)