{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T07:18:04Z","timestamp":1792653484792,"version":"4.5.0"},"reference-count":31,"publisher":"American Society for Microbiology","issue":"11","license":[{"start":{"date-parts":[[2025,11,12]],"date-time":"2025-11-12T00:00:00Z","timestamp":1762905600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,11,12]],"date-time":"2025-11-12T00:00:00Z","timestamp":1762905600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"Scientific Research Project of Beijing Youan Hospital, CCMU, 2024","award":["BJYAYY-YN2024-16"],"award-info":[{"award-number":["BJYAYY-YN2024-16"]}]},{"name":"Talent Cultivation Plan of Climbing the Peak of Beijing Municipal Hospital Administration","award":["DFL20221503"],"award-info":[{"award-number":["DFL20221503"]}]},{"name":"Beijing Natural Science Foundation Changping Innovation Joint Fund","award":["L234046"],"award-info":[{"award-number":["L234046"]}]},{"name":"Scientific Research Cultivation Fund of Capital Medical University","award":["PYZ24152"],"award-info":[{"award-number":["PYZ24152"]}]},{"name":"High\u2010Level Public Health Technical Talents Project of Beijing","award":["Subject Leaders\u201002\u201013"],"award-info":[{"award-number":["Subject Leaders\u201002\u201013"]}]},{"name":"Wang Baoen Liver Fibrosis Research Fund of China Foundation for Hepatitis Prevention and Control","award":["2025046"],"award-info":[{"award-number":["2025046"]}]},{"name":"R&DProgram of Beijing Municipal Education Commission","award":["KZ202010025035"],"award-info":[{"award-number":["KZ202010025035"]}]},{"name":"Chinese Institutes for Medical Research, Beijing","award":["CX24PY23"],"award-info":[{"award-number":["CX24PY23"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Clin Microbiol"],"published-print":{"date-parts":[[2025,11,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Timely and accurate diagnosis is essential for the effective treatment of\n                      <jats:italic toggle=\"yes\">Haemophilus influenzae<\/jats:italic>\n                      (HI) infection. Notably, a rapid point\u2010of-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\/\u03bcL 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.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Timely and accurate diagnosis is essential for the effective treatment of\n                        <jats:italic toggle=\"yes\">Haemophilus influenzae<\/jats:italic>\n                        (HI) infection. Notably, a rapid point\u2010of-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.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jcm.00535-25","type":"journal-article","created":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T13:23:55Z","timestamp":1758633835000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":9,"title":["An extraction-free, lyophilized one-pot RAA-CRISPR assay for point-of-care testing of\n                    <i>Haemophilus influenzae<\/i>"],"prefix":"10.1128","volume":"63","author":[{"given":"Yaling","family":"Cao","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/013xs5b60","id-type":"ROR","asserted-by":"publisher"}],"name":"Beijing Institute of Hepatology\/Beijing Youan Hospital, Capital 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