Highly specific detection of Neisseria gonorrhoeae based on recombinase polymerase amplification-initiated strand displacement amplification

Abstract

Neisseria gonorrhoeae is the only pathogen that causes gonorrhea, and can have serious consequences if left untreated. A simple and accurate detection method for N. gonorrhoeae is essential for the diagnosis of gonorrhea and the appropriate prescription of antibiotics. The application of isothermal recombinase polymerase amplification (RPA) to detect this pathogen is advantageous because of its rapid performance, high sensitivity, and minimal dependency on equipment. However, this simplicity is offset by the risk of false-positive signals from primer-dimers and primer-probe dimers. In this study, RPA-initiated strand displacement amplification (SDA) was established for the detection of N. gonorrhoeae, and eliminated false-positive signals from primer-dimers and primer-probe dimers. The developed biosensor allows for the reduced generation of nonspecific RPA amplification through the design of enzyme cleavage sites on primers, introduction of SDA, and detection of the final product using a molecular beacon (MB). Using this system, the DNA double strand is transformed into single-stranded DNA following SDA, thereby providing a more suitable binding substrate and improving the efficiency of MB detection. Amplification can be conducted below 37 °C, and the process can be completed within 90 min. The limit of detection was determined to be 0.81 copies/μL. This system is highly specific for N. gonorrhoeae and exhibits no cross-reactivity with other common urogenital pathogens. The results of this study are consistent with those of real-time PCR performed on clinical specimens of urogenital secretions. In summary, the biosensor is a simple and specific detection method for N. gonorrhoeae.

Keywords: Molecular beacon; Neisseria gonorrhoeae; Recombinase polymerase amplification; Strand displacement amplification.