Quantum dots photobleaching-based monochrome multiplexing in loop-mediated isothermal amplification detection of...

Multiplex nucleic acid amplification is essential for saving sample material and reducing reagent consumption. Common detection methods usually involve the use of fluorogenic probes that emit at different wavelengths, and the number of fluorescent channels often limits the degree of multiplexing. Photobleaching enables multiplex detection under a single excitation wavelength and emission wavelength, thereby overcoming the limitations inherent in traditional fluorescence detection methods. However, most reports rely on organic fluorescent dyes, and photostable quantum dots have often been neglected in photobleaching-based multiplexing. Herein, we present a fast and simple technique that enables monochrome multiplexed detection in loop-mediated isothermal amplification using quantum dot-525 nm and FAM fluorescent labels with the same green emission. This technique involves the inclusion of two fluorescent label primers and a quencher probe. If the target is present, the primer will emit the same fluorescent signal. However, with unamplified primers, the fluorescence is quenched. The two fluorescent signals exhibit different fluorescence changes during photobleaching, thereby distinguishing between different products. We successfully applied this method to loop-mediated isothermal amplification for S. aureus and L. monocytogenes. The method demonstrates high sensitivity and specificity and can be analyzed in complex biological samples. Additionally, a 2-plex loop-mediated isothermal amplification reaction in a single color channel was demonstrated. In the future, the color of quantum dots and the melting temperature of extension primers can also serve as independent parameters for detection. The integration of these two parameters may further improve the degree of multiplexing. This method could also be applied to rapid field-based screening.