Si trova su / Altri legami
© 2021 American Chemical Society.Loop–mediated isothermal amplification (LAMP) has been increasingly applied in nucleic acid detection for clinical diagnosis and monitoring pathogenic microorganisms due to its isothermal nature and high sensitivity. However, the false–positive signal resulting from the non–specific amplification and the complexity of primer design are still technically challenging for wide applications. In this paper, we developed the CRISPR/Cas12a–assisted sequence–specific detection of LAMP products to eliminate the effect of non–specific amplification from primer dimers and spurious amplicons. Moreover, by designing a pair of target–specific stem–loop DNA probes, we greatly simplified the primer design for LAMP. The DNA probes could be ligated to form a double–stem–loop DNA template by the detected target, which initiated LAMP reaction and achieved one–nucleotide resolution due to the highly specific ligase reaction. Using microRNAs (miRNAs) as the model targets, the CRISPR/Cas12a–assisted ligation–initiated loop–mediated isothermal amplification (CAL–LAMP) can sensitively detect as low as 0.1 fM miRNAs with high specificity.
