A structure-optimized molecular beacon-based RPA assay for highly sensitive and rapid detection of Neisseria gonorrhoeae.

Early detection of Neisseria gonorrhoeae (NG) is key to controlling its transmission and ensuring prevention and treatment. Recombinase polymerase amplification (RPA), combined with traditional fluorescent probe assays, is a promising method for NG detection. However, these traditional probes require four types of modifications involving cumbersome modification processes and high synthesis costs, and additional nucleic acid endonucleases are also needed for the reaction. In this paper, we propose a structure-optimized molecular beacon (SOMB) for RPA fluorescence detection in NG. We systematically investigated the effects of the length of the MB stem, base number of target recognition, and degree of partial closure of target recognition on the signal-to-noise ratio of the sensors, ultimately obtaining a SOMB with a unique secondary structure. The SOMB-based RPA assay platform provides rapid detection within 30 min at 37 °C with high specificity and detection limits as low as 0.96 copies/μL. In the detection of clinical samples, this method has good consistency with real-time PCR. Unlike conventional fluorescent probes, the SOMB for RPA proposed in this study has a simple and flexible design, requires few modifications, and does not require nucleic acid endonucleases.