An extraction-free, lyophilized one-pot RAA-CRISPR assay for point-of-care testing of <i>Haemophilus influenzae</i>

Timely and accurate diagnosis is essential for the effective treatment of <i>Haemophilus influenzae</i> (HI) infection. Notably, a rapid point-of-care testing (POCT) method for the diagnosis of HI DNA is still lacking. The aim of this study was to establish a one-pot detection device for HI DNA detection using CRISPR/Cas13a that would be applicable for POCT. We established a two-step polymerase chain reaction (PCR)-CRISPR assay, a two-step recombinase-aided amplification (RAA)-CRISPR assay, and a one-pot RAA-CRISPR assay. Additionally, reagent lyophilization, rapid lysis technology, and a one-pot detection device were integrated to construct an extraction-free one-pot detection device, named EFORCA (extraction-free one-pot RAA-CRISPR/Cas13a assay). Validation was performed on 90 simulated samples and 77 clinical samples. The two-step PCR/RAA-CRISPR assay for HI DNA detection was established and optimized, with a detection limit of 1 copy/μL and 100% specificity. The sensitivity and specificity of the two-step PCR-CRISPR assay for the 90 simulated clinical samples were 96.7% and 95%, respectively, and for the 77 clinical samples, 97.5% and 100%, respectively. The one-pot RAA-CRISPR assay and the EFORCA detected 50 and 25 CFU/mL HI in bacterial suspensions within 30 min, respectively. The valuation results for 77 clinical samples demonstrated that the sensitivity of both methods was 95%, which was greater than the sensitivity of quantitative real-time PCR (qPCR) (92.5%) with the same specificity of 100%. We developed an extraction-free one-pot RAA-CRISPR/Cas13a assay for HI detection, which effectively performs a POCT test and is a valuable tool for the early detection and monitoring of HI infection.IMPORTANCETimely and accurate diagnosis is essential for the effective treatment of <i>Haemophilus influenzae</i> (HI) infection. Notably, a rapid point-of-care testing (POCT) method for the diagnosis of HI DNA is still lacking. In this study, sensitive two-step polymerase chain reaction (PCR)-CRISPR and recombinase-aided amplification (RAA)-CRISPR assays were developed, followed by the establishment of the one-pot RAA-CRISPR assay through integration of RAA, CRISPR, and rapid lysis for HI detection. Additionally, reagent lyophilization, rapid lysis technology, and a one-pot detection device were integrated to construct the extraction-free one-pot RAA-CRISPR/Cas13a assay (EFORCA). With its high sensitivity, specificity, rapid turnaround time, and operational simplicity, this assay shows great potential as a practical diagnostic tool for HI infection in small laboratory settings.