Malachite green as a dual visual/electrochemical indicator for loop-mediated isothermal amplification: Application to SARS-CoV-2 detection

Loop -mediated isothermal amplification (LAMP) has arisen as an outstanding molecular technique for the amplification of nucleic acids, becoming an important alternative to PCR due to its simplicity and high sensitivity. However, traditional visual readout used in LAMP only provides qualitative results and can thus not provide a precise quantification of the target DNA/RNA in the sample. To overcome this, electroactive reporters incorporated in the reaction mix can provide a subsequent quantitative result of the analysis. In this work, malachite green, a commonly used colorimetric LAMP indicator, is used as an electrochemical probe to obtain quantitative results. For this purpose, its optical and electrochemical behavior has been assessed for further use in LAMP detection. Using SARS-CoV-2 RNA as template, we have developed a quantitative detection method, proving the usefulness of this molecule as a dual optical/electrochemical indicator. Thereby, with the electrochemical detection using screen -printed electrodes on which a drop of the LAMP reaction is deposited, a calibration curve with a linear dynamic range comprised between 22 and 4296 copies/mu L was achieved, with a practical limit of detection of 22 copies/mu L. In this method, validated with clinical samples, malachite green is used, by the first time, as a dual visual electrochemical/visual probe that can provide both an initial visual qualitative assessment together with an electrochemical quantification.