Rapid Detection of DNA Methylation with a Novel Real-Time Fluorescence Recombinase-Aided Amplification Assay
Ziyu He, Zengrui Tong, Boyu Tan, Xuliang He, Tao Zhang, Yuan Guo, Lian Jin, Nongyue He, Song Li, Zhu Chen
Abstract
Researchers have conducted in-depth research on DNA methylation mechanism, which is related to various diseases such as deficiency of imprinted gene and occurrence of tumors. This study provides a novel rapid quantitative detection assay and real-time fluorescence recombinase-aided amplification assay (RAA) for DNA methylation. Firstly, specific sequence of methylation genes was chosen and primers and fluorogenic probe for RAA experiment were designed and synthesized. Lastly, these amplification products were proven by sequencing and analysis. Results showed that the amplification efficiency and template concentration of RAA had linear dependent ( R 2 > 95%) when the concentration range was 4.64×10 8 copies/ μ L˜4.64×10 4 copies/ μ L. The test assay can also detect positive samples when the template concentration is below 4.64×10 4 copies/ μ L. Remarkably, the entire experiment process only takes 15–20 minutes, so it is beneficial for rapid bedside simple screening of some special DNA methylation sites, such as detection of resistance genes. In a word, this method has very great potential for diseases with DNA methylation in clinical settings, especially if methylation analysis needs to be done quickly and easily.