CRISPR-Cas12a Assay for Rapid and Specific Detection of Shigella flexneri 2a in Clinical Samples

Shigella flexneri 2a is the most common cause of shigellosis, a major public health concern in developing countries. Rapid and reliable diagnostic tools are critical for timely outbreak detection and management. Leveraging Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, we developed a CRISPR-Cas12a-based assay for the rapid and specific detection of S. flexneri 2a and validated its performance using stool specimens from patients. Two guide RNAs targeting the gtrII and gtrX genes-unique markers of the S. flexneri 2a serotype-were designed to ensure specificity. Recombinase Polymerase Amplification (RPA) was coupled with Cas12a-mediated collateral cleavage for signal amplification, with detection by fluorescence or lateral flow. Analytical sensitivity, specificity, and clinical accuracy were compared with conventional PCR using purified DNA and 588 clinical stool specimens. The CRISPR-Cas12a assay achieved a detection limit of 10 copies/uL, comparable to PCR, and showed 100% analytical specificity without cross-reactivity to other bacteria. The isothermal reaction operated at room temperature and was completed within one hour. Both readouts allowed visual interpretation without specialized equipment. Clinical validation demonstrated a diagnostic sensitivity of 95% and specificity of 98%, confirming robust performance. This study provides two key advances: it establishes a CRISPR-Cas12a assay specifically targeting S. flexneri 2a, the predominant serotype, and validates it using a large clinical cohort. The assays simplicity, speed, and high diagnostic accuracy make it a valuable tool for clinical diagnostics and field-based surveillance in resource-limited settings. IMPORTANCERapid and accessible diagnostics are essential for effective management of infectious diseases such as shigellosis. We developed a CRISPR-Cas12a-based assay that specifically detects Shigella flexneri 2a, the predominant serotype responsible for the global disease burden. This assay integrates isothermal amplification with CRISPR-mediated detection to achieve single-copy sensitivity within one hour, eliminating the need for complex instrumentation. Dual fluorescence and lateral-flow readouts enable flexible use in both clinical laboratories and low-resource settings. The methods simplicity, accuracy, and adaptability demonstrate the practical potential of CRISPR diagnostics for point-of-care applications. By enabling rapid, on-site identification of S. flexneri 2a, this approach can significantly improve clinical diagnosis and strengthen public health responses to enteric pathogen outbreaks.