Ultrasensitive and Quantitative DNA Methylation Detection Method Based on the MutS Protein

DNA methylation is closely related to cancer. It is generally accepted that DNA methylation detection is crucial in cancer diagnosis, prognosis, and treatment monitoring. Therefore, there is an urgent demand for developing a simple, rapid, highly sensitive, and highly specific methylation detection method to detect DNA methylation at specific sites quantitatively. In this work, we introduce a DNA methylation detection method based on MutS and methylation-specific PCR, named MutS-based methylation-specific PCR (MB-MSP), which has the advantages of simplicity, speed, high specificity, sensitivity, and broad applicability. Utilizing the MutS's ability to bind mismatched base pairs, we inhibit not only the amplification of unmethylated DNA but also nonspecific primer amplification. We achieved a detection sensitivity of 0.5% for the methylated genes of ACP1, CLEC11A, and SEPT9 by MB-MSP. It has a good linear relationship and a detection time of only 1.5 h. To validate the feasibility of the MB-MSP method in clinical application, we conducted methylation detection on plasma-circulating tumor DNA samples from 10 liver cancer patients and 5 healthy people, achieving a 100% accuracy rate. In conclusion, MB-MSP, as a novel and reliable DNA methylation detection tool, holds significant application value and potential for advancing early cancer diagnosis.