Rapid and sensitive detection of Haemophilus influenzae using multiple cross displacement amplification combined with CRISPR-Cas12a-based biosensing system

<i>Haemophilus influenzae</i> (<i>H. influenzae</i>, Hi) is an opportunistic bacterium that colonizes the upper respiratory tract of humans and frequently causes meningitis, pneumonia, sepsis, and other severe infections in children. Early and accurate detection of <i>H. influenzae</i> is essential for effective diagnosis and treatment. In this study, we established a novel diagnostic method by integrating the CRISPR-Cas12a detection platform with multiple cross-displacement amplification (MCDA), termed the Hi-MCDA-CRISPR assay. This method offers an efficient and highly precise diagnostic tool for the identification of <i>H. influenzae</i>. In the Hi-MCDA-CRISPR system, the outer membrane protein (OMP) <i>P6</i> of <i>H. influenzae</i> was pre-amplified using the MCDA assay. The CRISPR-Cas12a-gRNA complex specifically recognized and bound to the amplified gene, forming a ternary complex that triggered the nonspecific trans-cleavage of the Cas12a effector, which subsequently degraded the fluorescent-quenched single-stranded DNA (ssDNA) probes, resulting in the emission of detectable fluorescent signals. After optimizing the reaction conditions, the Hi-MCDA-CRISPR assay proved capable of completing <i>H. influenzae</i> detection within 45 min, including a 40 min MCDA pre-amplification at 62 °C and a 5 min CRISPR-Cas12a cleavage at 37 °C. The assay was able to detect <i>H. influenzae</i> genomic DNA at concentrations as low as 50 fg and showed no cross-reactivity with non-<i>H. influenzae</i> pathogens. Furthermore, the Hi-MCDA-CRISPR assay successfully analyzed 65 clinical sputum samples. These findings suggest that the Hi-MCDA-CRISPR assay is a promising new detection tool for the rapid and reliable diagnosis of <i>H. influenzae</i> infection.