Multiple Cross Displacement Amplification Combined with Real-Time Fluorescence Monitoring for Efficient, Specific,...

Invasive meningococcal disease, caused by Neisseria meningitidis (N. meningitidis), is a critical global health issue, necessitating swift and precise diagnostics for effective management and control. Here, we introduce a novel diagnostic assay, NM-RT-MCDA, that combines multiple cross displacement amplification (MCDA) with real-time fluorescence detection, targeting a specific ctrA gene region in the N. meningitidis genome. The assay utilizes a primer set designed for high specificity and incorporates a fluorophore-quencher pair with a restriction endonuclease site for real-time monitoring. Optimized at 65 °C for 40 min, NM-RT-MCDA demonstrates exceptional specificity, with no cross-reactivity observed with nontarget species. It achieves a remarkable sensitivity, detecting as low as 100 fg of genomic DNA per reaction, and has been successfully applied to clinical sputum samples, matching the sensitivity of nanoparticle-based lateral flow biosensors. The assay's rapid turnaround time, completed within an hour including DNA extraction and amplification, positions NM-RT-MCDA as a promising diagnostic tool for various clinical scenarios, potentially facilitating timely diagnosis and intervention in invasive meningococcal disease management.