A facile cascade signal amplification strategy using DNAzyme loop-mediated isothermal amplification for the ultrasensitive colorimetric detection of Salmonella

摘要

To make the direct readout of loop-mediated isothermal amplification (LAMP) simpler and more reliable and to enhance the visual sensitivy of LAMP detection, we developed a facile cascade signal-on colorimetric DNAzyme LAMP (dLAMP) sensor that integrates the amplification power of LAMP and the inherent catalytic activity of the DNAzyme for the simple and ultrasensitive analysis of targets. First, a signal inner primer with a 17-nt DNAzyme complementary sequence (a signal precursor) in its middle insertable region was asymmetrically designed for the next amplification and colorimetric reaction. In the presence of target DNA, the target specifically initiated LAMP, and the signal precursor was amplified to a larger number of DNAzyme sequences during the target DNA amplification. With the addition of hemin, free DNAzyme fragments, which were released by ultrasonication and pPNA, formed G-quadruplex-hemin conjugates that act as a signal readouts for the direct colorimetric assay. Even less 0.5 pg genomic DNA were detected using the established one-step cascade signal amplification strategy without the need for gels or other apparatus. Moreover, the novel colorimetric strategy showed high fidelity in DNA detection and visual sensitivity of LAMP. This method may pave the way for the point-of-care detection and other PCR-based colorimetric analyses.