Hybridization chain reaction-assisted CRISPR/Cas12a strategy for rapid and visual detection of Haemophilus influenzae.

Haemophilus influenzae (H. influenzae) is a major pathogen causing community-acquired pneumonia in children, posing a serious threat to children's health. Rapid and convenient testing is needed for effective treatment. Traditional detection methods, such as bacterial culture and qPCR are cumbersome to operate, and require sophisticated instrumentation. Here, we developed a visual CRISPR/Cas detection platform that integrates the hybridization chain reaction (HCR) and horseradish peroxidase (HRP)-catalyzed 3,3',5,5'-Tetramethylbenzidine (TMB) colorimetric change, called Vi-CasHCP. The single-base recognition capability of CRISPR/Cas12a improves the specificity, while the efficiency of HCR shortens the detection time and improves the sensitivity. The peroxidase-like activity of HRP catalyzes the oxidation of TMB to oxTMB, resulting in a blue color change. Vi-CasHCP achieved a detection limit of 11.8 CFU/mL in 70 min, after Recombinase polymerase amplification. Clinical validation using 50 respiratory samples showed complete diagnostic agreement with qPCR, with 100% sensitivity, specificity, PPV, and NPV. Therefore, this platform offers rapid detection, requires no complex instruments, and has high sensitivity, providing fast and accurate diagnostic support for clinical practice, and is particularly suitable for resource-limited settings.