An amplification-free detection method of nucleic acids by a molecular beacon probe based on endonuclease activity

Abstract

Sensitive and specific identification of pathogen is in great demand but remains challenging. Herein, we developed an endonuclease dependence molecular beacon assay (DEMBA). We firstly labeled the DEMBA probes by a 6-Carboxyfluorescein at the 5’-end as the fluorophore and a black hole quencher at the 3’-end. Then we recorded the fluorescence signals for analyzation. In addition, the lateral flow dipstick (LFD) DEMBA probes are labeled with fluorescein isothiocyanate (FITC) at the 5’‑end, and biotin at the 3’-end. The digested LFD DEMBA probes can be detected by visual inspection of a colorimetric signal on a lateral flow dipstick. In fluorescence method, we detected ssDNA molecules down to 1 pM and ssRNA to 10 pM within 10 min. Meanwhile, in lateral flow dipstick method, the ssDNA and ssRNA molecules detection limits are 10 pM. Successfully, we also detected the single nucleotide spot mutation (SNP) without false positive or false negative interpretation. Furthermore, the DEMBA is an amplification-free method, which can avoid the aerosol pollution and be easy-to-perform. In conclusion, the DEMBA is an isothermal detection method with simple design, high sensitivity, wide-ranged application, easy operation and time saving.