A dual-readout RAA-CRISPR/Cas13a diagnostic platform for rapid and sensitive detection of Eggerthella lenta

Eggerthella lenta (E. lenta) is an opportunistic anaerobic pathogen associated with severe systemic infections, yet rapid and accurate diagnostic tools remain limited. To address this challenge, we developed a highly sensitive and specific dual-readout diagnostic platform integrating recombinase-aided amplification (RAA) with the CRISPR/Cas13a system, targeting the highly conserved rsmG gene of E. lenta. The assay offers two detection modalities: a real-time fluorescence readout and a visually interpretable lateral flow strip. Analytical evaluation demonstrated that the fluorescence-based assay achieved a limit of detection (LOD) of 4.4 copies per reaction (95% CI: 3.7-5.6 copies/reaction), while the instrument-free lateral flow assay yielded an LOD of 10⁴ copies per reaction. The platform exhibited exceptional specificity, showing no cross-reactivity with 10 common non-target bacterial species. Clinical validation was performed using 24 synovial fluid samples, all confirmed positive for E. lenta by Sanger sequencing. The fluorescence assay successfully detected all 24 samples, achieving a detection rate of 100% (24/24). In parallel, the lateral flow assay detected 21 of the 24 positive samples, yielding a detection rate of 87.5% (21/24). The three samples undetected by the lateral flow strip were verified as true positives by sequencing, indicating that the discrepancy was due to the lower analytical sensitivity of the strip format rather than a lack of specificity. In conclusion, this dual-mode RAA-CRISPR/Cas13a platform serves as a robust and practical tool for rapid clinical diagnosis and point-of-care (POC) triaging of E. lenta infections. The fluorescence format is optimal for high-sensitivity laboratory testing, whereas the lateral flow variant provides a deployable alternative for rapid, point-of-care screening in resource-limited environments.