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Although real-time quantitative polymerase chain reaction (RT-qPCR) remains the gold standard for HR-HPV detection, its dependence on sophisticated equipment, complex procedures, and trained personnel limits accessibility. Here, we developed a simplified assay for 14 HR-HPV types by integrating direct lysis, enzyme-mediated isothermal rapid amplification (ERA), and CRISPR-Cas12a-mediated cleavage into a streamlined workflow that requires only a basic isothermal heating device. The optimized system achieved a sensitivity of 50 copies per reaction with no cross-reactivity, while a refined lysis buffer containing 20% Chelex-100 minimized inhibition from vaginal swab samples, thereby enhancing detection performance. Validation with 152 clinical samples demonstrated 97.62% sensitivity and 100% specificity, confirming the reliability of the method. This user-friendly and cost-effective assay requires minimal equipment, enabling rapid and field-deployable HR-HPV detection, and offers a practical alternative to conventional laboratory-based approaches, particularly in resource-limited settings.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>High-risk human papillomavirus (HR-HPV) is the principal etiological agent of cervical cancer, and early detection remains central to effective disease prevention. Current PCR-based assays, however, rely on specialized laboratories and trained personnel, limiting their deployment in many settings. Here, we report a streamlined CRISPR-Cas12a assay that integrates direct sample lysis, ERA, and CRISPR-based detection into a single workflow operable with only a simple heating device to determine the presence of 14 HR-HPV types. The assay achieves high analytical sensitivity, strong specificity, and robust clinical performance while maintaining low cost and ease of use. This platform enables rapid HR-HPV detection and scalable screening, particularly in resource-constrained environments, with the potential to facilitate earlier intervention and reduce cervical cancer incidence.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.03036-25","type":"journal-article","created":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T12:34:16Z","timestamp":1766752456000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Development and clinical validation of an ERA-CRISPR\/Cas12a assay for the rapid detection of 14 high-risk HPV types"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4619-0543","authenticated-orcid":true,"given":"Zhijie","family":"Wang","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00p991c53","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/00p991c53","id-type":"ROR","asserted-by":"publisher"}],"name":"National Clinical Research Center for Obstetrics and Gynecology, Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","place":["Wuhan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ting","family":"Hu","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00p991c53","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/00p991c53","id-type":"ROR","asserted-by":"publisher"}],"name":"National Clinical Research Center for Obstetrics and Gynecology, Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","place":["Wuhan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanxin","family":"Liu","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00p991c53","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/00p991c53","id-type":"ROR","asserted-by":"publisher"}],"name":"National Clinical Research Center for Obstetrics and Gynecology, Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","place":["Wuhan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hu","family":"Zhou","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00p991c53","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/00p991c53","id-type":"ROR","asserted-by":"publisher"}],"name":"National Clinical Research Center for Obstetrics and Gynecology, Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","place":["Wuhan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyi","family":"Lv","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00p991c53","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","place":["Wuhan, China"]},{"id":[{"id":"https:\/\/ror.org\/00p991c53","id-type":"ROR","asserted-by":"publisher"}],"name":"National Clinical Research Center for Obstetrics and Gynecology, Cancer Biology Research Center (Key Laboratory of the Ministry of Education), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology","place":["Wuhan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Li","sequence":"additional","affiliation":[{"name":"Wuhan Kandwise Biotechnology, Inc. 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