A one-step LAMP-CRISPR/Cas12a platform targeting lytA and cpsA for rapid parallel identification and virulence...

Streptococcus pneumoniae (S. pneumoniae, SPN) is the primary pathogen causing community-acquired pneumonia (CAP), purulent meningitis, sepsis, and acute otitis media. Accurate species identification and rapid differentiation of invasive strains are critical for clinical management and infection control. Based on loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a technologies, this study established a parallel dual-target detection platform targeting the S. pneumoniae-specific lytA gene and the capsular polysaccharide synthesis regulatory gene cpsA. A dual-readout mode combining fluorescence detection (FD) and lateral flow test strip (LFTS) was adopted to meet diverse laboratory testing requirements. For each target-specific reaction, LAMP amplification and CRISPR/Cas12a cleavage were sequentially performed in a single closed tube using paraffin-oil stratification to suppress solvent evaporation, effectively preventing aerosol contamination. Range-finding serial-dilution assays revealed that the lowest gDNA concentrations producing positive signals in all three technical replicates reached 1 fg μL^-1 (FD) and 10 fg μL^-1 (LFTS). This assay specifically distinguished S. pneumoniae from other common clinical pathogens and preliminarily identified encapsulated invasive strains, with 100% sensitivity and specificity consistent with conventional PCR. This LAMP-CRISPR/Cas12a platform, with its closed-tube design, avoids contamination, while FD and LFTS dual readouts cater to diverse testing needs. Featuring high sensitivity, excellent specificity, simplicity, and speed, it provides an innovative and practical solution for the rapid diagnosis of S. pneumoniae and holds great clinical potential.