A Rapid and Specific RAA-CRISPR/Cas13a Assay for Detecting Streptococcus mutans.

Abstract

Streptococcus mutans is recognized as a major etiological agent of dental caries, a highly prevalent chronic global health issue. The early identification of such cariogenic pathogens is therefore critical for implementing effective preventive strategies. To address this need, we established an integrated detection platform coupling recombinase-aided amplification (RAA) with CRISPR-Cas13a for S. mutans detection and evaluated its performance against conventional polymerase chain reaction (PCR). The proposed RAA-CRISPR-Cas13a assay exhibited superior sensitivity and faster turnaround time compared to PCR. A distinctive feature of the assay design was the inclusion of a T7 promoter sequence within the forward primer, thereby facilitating direct in vitro transcription without requiring an additional oligonucleotide. The assay demonstrated a broad quantitative detection range from 10¹ to 10⁹ CFU/mL, with a limit of detection (LOD) reaching 3.2 CFU/mL. A strong linear relationship (R² > 0.98) was established between the fluorescence response and the logarithmic bacterial concentration, underscoring the assay's accuracy and robustness. This sensitive and rapid detection system shows considerable promise for screening high-risk populations, allowing not only early detection but also potential evaluation of caries severity and progression. Its simplicity and minimal instrumentation requirements make it particularly suitable for low-resource healthcare environments.