Sensitive detection of methylated DNA using the short linear quencher-fluorophore probe and two-stage isothermal amplification assay

DNA methylation is an important epigenetic modification of genomes and is associated with various human diseases. Here, we develop a sensitive approach for DNA methylation assay using the short linear quencher-fluorophore DNA probe (QF probe) and two-stage isothermal amplification. With bisulfite treatment, the methylated DNA target is able to hybridize with the template to initiate the strand displacement amplification (SDA), generating abundant triggers which can further hybridize with the QF probes to form the DNA duplexes. The subsequent recognition of DNA duplexes and the cleavage of QF probes by the nicking enzyme can initiate the nicking enzyme signal amplification (NESA), inducing a significant fluorescence enhancement. While in the absence of methylated DNA, neither SDA nor NESA reaction can be initiated and no fluorescence enhancement is observed. This method exhibits high sensitivity with a detection limit of 0.78 pM, and can distinguish as low as 0.1% methylation level from the mixture of methylated and unmethylated DNA. Notably, the introduction of SDA into NESA can improve the detection sensitivity by up to 2 orders of magnitude as compared with the NESA assay, and it can even discriminate single-base mismatched methylated DNA.