An APE1-mediated isothermal target cycling amplification for label-free and rapid detection of miRNA

Developing simple, rapid and sensitive strategies for miRNA analysis is extremely important for diseases early diagnosis. Herein, an APE1-mediated isothermal target cycling amplification system combined with magnetic separation was established for label-free and rapid detection of miRNA. As a proof-of-concept, miR-1246 was selected as research model. In the detection system, when the miR-1246 is present, it hybridizes with AP probes of AP-MBs to form a double-stranded, in which the APE1 recognizes specific AP site of double-stranded and cleaves the AP probes, releasing a sequence containing a G-quadruplex (G4). After the cleavage, target miR-1246 can be released to hybridize with another AP of AP-MBs, triggering a continues cleavage reaction. After magnetic separation, label-free analysis of miRNA was achieved by measuring the fluorescence of G4/ThT. Due to the high efficiency and specificity of APE1 toward AP sites in dsDNAs, this method exhibits high specificity and sensitivity with the capability of distinguishing miRNAs with single-base mismatch. The system could detect miRNA with high sensitivity within 2 h, and the limit of detection (LOD) was calculated as 25.6 fM. Furthermore, high accuracy has been achieved through recovery experiments and successful attempts has been made in applying the approach to detect miR-1246 in serum samples from breast cancer patients and normal people. This method is expected to be an effective tool for miRNA-related research and clinical early diagnosis.

Automated summary

This study developed a highly efficient and sensitive miRNA detection method by combining APE1-mediated isothermal target cycling amplification system with magnetic separation. The label-free and rapid detection of miRNA was achieved through the recognition and cleavage of AP probes by APE1, and the fluorescence measurement of G4/ThT. The method showed high specificity and sensitivity, and demonstrated its application in detecting miR-1246 in serum samples from breast cancer patients and normal people.