biostudies-literatureCao YR&DProgram of Beijing Municipal Education CommissionTalent Cultivation Plan of Climbing the Peak of Beijing Municipal Hospital AdministrationChinese Institutes for Medical Research, BeijingBeijing Natural Science Foundation Changping Innovation Joint FundWang Baoen Liver Fibrosis Research Fund of China Foundation for Hepatitis Prevention and ControlHigh-Level Public Health Technical Talents Project of BeijingScientific Research Cultivation Fund of Capital Medical UniversityScientific Research Project of Beijing Youan Hospital, CCMU, 2024e0053525https://www.ebi.ac.uk/biostudies/studies/S-EPMC12607747biostudies-literatureUnknown63(11)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 Journal of clinical microbiologyAn extraction-free, lyophilized one-pot RAA-CRISPR assay for point-of-care testing of &lt;i&gt;Haemophilus influenzae&lt;/i&gt;.PMC12607747BJYAYY-YN2024-16CX24PY23KZ2020100250352025046Subject Leaders-02-13L234046DFL20221503PYZ24152Xu LPan ZRen FFan ZHuang JCao YYang QWang JMo YfalseAn extraction-free, lyophilized one-pot RAA-CRISPR assay for point-of-care testing of &lt;i&gt;Haemophilus influenzae&lt;/i&gt;.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 2025-01-01T00:00:00Z2025 Nov2026-06-05T13:22:41.382Z2026-06-05T03:05:56.655ZS-EPMC126077474098581210.1128/jcm.00535-25