OBJECTIVE: Mycoplasma pneumoniae (MP) is a leading cause of community-acquired pneumonia in children. Conventional detection methods often lack the speed or accessibility required for point-of-care testing (POCT). This study aims to develop a rapid, highly sensitive, and instrument-free diagnostic platform integrating Enzymatic Rapid Amplification (ERA), CRISPR/Cas12a cleavage, and Lateral Flow Assay (LFA) for visual MP detection.
METHODS: Specific primers targeting the conserved P1 adhesin gene of MP were screened for ERA efficiency. A CRISPR/Cas12a system was designed to specifically recognize the ERA amplicons, triggering the trans-cleavage of a reporter probe. The results were visually interpreted using LFA strips. The assay's limit of detection (LoD), specificity against six common respiratory pathogens, and clinical performance on 80 throat swab samples were evaluated.
RESULTS: The optimized ERA-CRISPR/Cas12a-LFA assay can be completed within 40 minutes. The LoD was established at 200 copies/mL. Specificity testing showed no cross-reactivity with S. pneumoniae, H. influenzae, or other tested pathogens. In clinical validation (n = 80), the assay demonstrated a sensitivity of 96.23% and specificity of 100% compared to qPCR, with a Kappa value of 0.945.
CONCLUSION: The established ERA-CRISPR/Cas12a-LFA method offers a rapid, sensitive, and specific alternative for MP screening. Its minimal equipment requirements make it highly suitable for resource-limited settings and primary care clinics.
