Development of a Rapid and Efficient RPA-CRISPR/Cas12a Assay for Mycoplasma pneumoniae Detection

Abstract

Mycoplasma pneumoniae (MP) is a one of most common pathogen in causing respiratory infection in children and adolescents. Rapid and efficient diagnostic methods are crucial for control and treatment of MP infections. Herein, we present an operationally simple, rapid and efficient molecular method for MP identification, which eliminates expensive instruments and specialized personnel. The method combines recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) 12a-based detection, with an optimal procedure less than 1 h from sample to result including DNA extraction (25 min), RPA reaction (39°C for 15-20 min), CRISPR/Cas12a detection (37°C for 10 min) and visual detection by naked eyes (2 min). This diagnostic method shows high sensitivity (two copies per reaction) and no cross-reactivity against other common pathogenic bacteria. Preliminary evaluation using 201 clinical samples shows sensitivity of 99.1% (107/108), specificity of 100% (93/93) and consistency of 99.5% (200/201), compared with real-time PCR method. The above data demonstrate that our developed method is reliable for rapid diagnosis of MP. In conclusion, the RPA-CRISPR/Cas12a has a great potential to be as a useful tool for reliable and quick diagnosis of MP infection, especially in primary hospitals with limited conditions.

Keywords: CRISPR/Cas12a; Mycoplasma pneumoniae; RPA; recombinase polymerase amplification; visual detection.

FIGURE 1

Schematic illustration of RPA-CRISPR/Cas12a assay for detection of MP. RT, room temperature.

FIGURE 2

Time optimization for CRISPR/Cas12a assay. CRISPR/Cas12a assay is performed at varying times from 5 to 20 min, and the fluorescence signals are directly observed by naked eyes under blue light. The RPA products are obtained from 2 copies genome. NC, negative control.

FIGURE 3

Sensitivity confirmation of RPA-CRISPR/Cas12a assay for MP detection. (A) Direct observation by naked eyes under blue light. (B) Visualization of RPA products by agarose gel electrophoresis. (C) Real-time fluorescence applied for further confirming the results. Tube/signal 1-8 represent template level (genomic DNA of M129) of 2 × 10⁶, 2 × 10⁵, 2 × 10⁴, 2 × 10³, 2 × 10², 2 × 10¹, 2 × 10⁰, 2 × 10^–1 copies per reaction. NC, negative control.

FIGURE 4

Analytical specificity of RPA-CRISPR/Cas12a assay for MP detection. The RPA-CRISPR/Cas12a assay is conducted using genomic DNA extracted from 19 pathogens. Tube 1-5, clinical isolates of MP; Tube 6-19, Klebsiella pneumonia ATCC 10031, Klebsiella pneumonia ATCC 700603, Klebsiella pneumonia isolated strain, Haemophilus influenzae isolated strain, Acinetobacter baumannii ATCC 19606, Acinetobacter baumannii isolated strain, Streptococcus pneumoniae isolated strain, Staphylococcus aureus isolated strain, Staphylococcus aureus isolated strain, Pseudomonas aeruginosa isolated strain, Group B Streptococcus isolated strain, isolated strain of carbapenem-resistant Enterobacter sp., Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212; PC, positive control; NC, negative control.