A one-pot CRISPR/Cas12b assay for extraction-free and visual detection of Haemophilus influenzae

Haemophilus influenzae is a major respiratory pathogen, particularly in pediatric populations, and its rapid and accurate detection is critical for early diagnosis and targeted treatment. Traditional diagnostic methods, such as bacterial culture and PCR, are often time-consuming and require specialized equipment. In this study, we developed the Hi-ExCad assay, a one-pot, extraction-free CRISPR/Cas12b-based system for the rapid, simple, and accurate detection of H. influenzae. The assay integrates loop-mediated isothermal amplification and CRISPR/Cas12b detection in a single reaction, enabling direct detection from clinical sputum samples without the need for nucleic acid extraction. The Hi-ExCad assay demonstrated high specificity, correctly identifying H. influenzae in clinical samples, with a limit of detection of 1 pg of genomic DNA. The assay also exhibited excellent performance in real-time detection and visual result interpretation under blue light, making it highly suitable for point-of-care testing and resource-limited settings. These findings suggest that the Hi-ExCad assay provides a rapid, reliable, and user-friendly method for the detection of H. influenzae, with significant potential for clinical application.