{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T23:15:40Z","timestamp":1788563740353,"version":"build-2803163510"},"reference-count":49,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T00:00:00Z","timestamp":1759881600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T00:00:00Z","timestamp":1759881600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100017531","name":"Medical and Health Research Project of Zhejiang Province","doi-asserted-by":"publisher","award":["2024KY1444"],"award-info":[{"award-number":["2024KY1444"]}],"id":[{"id":"10.13039\/501100017531","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Zhejiang Provincial Natural Science Foundation of China","award":["LTGC23H200004"],"award-info":[{"award-number":["LTGC23H200004"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Clin Microbiol"],"published-print":{"date-parts":[[2025,10,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Group B\n                      <jats:italic toggle=\"yes\">Streptococcus<\/jats:italic>\n                      (GBS) is a major cause of neonatal infections, and rapid detection is essential for timely clinical intervention. In this study, we developed an extraction-free, one-pot CRISPR\/Cas12b assay for visual detection of GBS by combining with isothermal amplification, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). The results showed that LAMP-CRISPR\/Cas12b outperformed RPA-CRISPR\/Cas12b system across all template concentrations, especially in low-copy template (30 and 10 copies\/test) detection. To enhance the detection performance of RPA-CRISPR\/Cas12b, we introduced a two-temperature protocol, with RPA reaction at 39\u00b0C followed by Cas12b activation at 62\u00b0C. Through the two-temperature approach, the detection rate of RPA-CRISPR\/Cas12b system was significantly improved even in low-copy samples, achieving a sensitivity of 10 copies\/test (1 copy\/\u03bcL). Clinical validation using 60 vaginal-rectal swab samples showed 96.7% and 98.3% of concordance when compared to culture and qPCR methods, respectively. This assay offers a rapid (&lt;1 h), highly sensitive, and user-friendly solution without requiring nucleic acid extraction or sophisticated instruments. Its compatibility with visual signal detection makes it ideal for point-of-care testing, especially in low-resource or time-sensitive settings. The platform can be adapted for broader pathogen detection in future field diagnostics.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      This study presents a rapid, convenient, and highly accurate method for Group B\n                      <jats:italic toggle=\"yes\">Streptococcus<\/jats:italic>\n                      (GBS) detection by integrating the CRISPR\/Cas12b system with recombinase polymerase amplification, an isothermal nucleic acid amplification technique. To streamline the workflow, we established a one-pot, extraction-free assay that significantly reduces the detection time. Through the systematic optimization of the dual-temperature conditions, we enhanced the amplification efficiency of target DNA, thereby improving the sensitivity of the CRISPR\/Cas12b system. Additionally, the incorporation of a UV-visible detection system enables visual readout, facilitating instrument-free testing suitable for point-of-care (POC) applications.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jcm.00819-25","type":"journal-article","created":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T13:01:51Z","timestamp":1758286911000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":9,"title":["An extraction-free and one-pot two-temperature CRISPR\/Cas12b system for visual detection of Group B\n                    <i>Streptococcus<\/i>\n                    by integrating with RPA"],"prefix":"10.1128","volume":"63","author":[{"given":"Chengman","family":"Zhao","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/014v1mr15","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Internal Medicine, Affiliated Xiaoshan Hospital, Hangzhou 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