A Rapid Detection Method for Mycoplasma Pneumoniae through the Combination of Recombinase Polymerase Amplification and CRISPR-Cas12a

Abstract

Background: There is a primary pathogen responsible for Mycoplasma pneumoniae pneumonia (MPP) in children called Mycoplasma pneumoniae (MP). Patients with mild symptoms usually complain of paroxysmal dry cough by stimulation and fever, while those with severe conditions may develop bronchopneumonia, pleural effusions, and lung abscesses, and even accompanied by lesions in the nervous system or urinary system. We aimed to develop a rapid detection method for MP adhesion protein P1 by merging recombinase polymerase amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats/associated proteins (CRISPR/Cas12a) gene editing. Methods: In this study, the sequence of P1 adhesion protein region (GenBank: AF290001.1) of MP was obtained from the national center of biotechnology information (NCBI) database. Five groups of primers and probes were designed using Primer Premier 5 software (Premier Canada Inc., Charlotte, NC, USA), and the best primers were selected. Furthermore, in the meantime, the corresponding real-time fluorescence quantitative polymerase chain reaction (RT-PCR) primers were designed for subsequent validation tests to determine the reagent proportion, reaction time, reaction temperature, and primer-probe concentration of the reaction system. Finally, diseased clinical samples (strong positive, medium positive, weak positive) and healthy clinical samples (1, 2, 3) were used to verify the optimized cutting system. Results: Through the experiments, we verified that the optimal reaction time for RPA at 37 °C was 30 min, and the best product was obtained by cleaving Cas12a-crRNA for 60 min, with a result fetched within 2 h. Conclusions: This method is not only fast and convenient in operation but also ensures the specificity, rapid efficiency and visualization of the test results without relying on instruments and professional personnel. It provides a solution to the subjective judgment of the culture test method and the difficulty of smear preparation.