{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T15:47:12Z","timestamp":1789660032197,"version":"4.0.1"},"reference-count":35,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T00:00:00Z","timestamp":1779148800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Objectives<\/jats:title>\n                    <jats:p>\n                      To address the unmet need for rapid, accurate diagnosis of\n                      <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                      (MTB) and multidrug-resistant tuberculosis (MDR-TB), we developed and validated a clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas)-based diagnostic assay.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      A multiplex-recombinase polymerase amplification (RPA) coupled CRISPR-\n                      <jats:italic>Alicyclobacillus acidiphilus<\/jats:italic>\n                      Cas12b (AaCas12b) assay was established for simultaneous detection of MTB by targeting the specific insertion sequence\n                      <jats:italic>IS6110<\/jats:italic>\n                      and the two most common drug resistance mutations,\n                      <jats:italic>rpoB<\/jats:italic>\n                      1349C&amp;gt;T for rifampicin resistance and\n                      <jats:italic>katG<\/jats:italic>\n                      944G&amp;gt;C for isoniazid resistance. The assay supported dual-readout signal detection using both a fluorescent platform and lateral flow chromatography (LFC). Its diagnostic performance was evaluated in 48 clinical samples using WHO-recommended GeneXpert MTB\/RIF, phenotypic drug susceptibility testing (pDST), and sequencing as reference standards.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The multiplex-RPA CRISPR-AaCas12b assay showed a limit of detection (LoD) of 1.5 CFU\/mL for MTB detection, with a sensitivity of 97.1% and a specificity of 100% using culture as the reference standard, and a total turnaround time of 30\u00a0min (20\u00a0min for RPA and 10\u00a0min for CRISPR cleavage). For MDR-TB-related mutations, the assay achieved a sensitivity of 94.1% and a specificity of 100% for\n                      <jats:italic>rpoB<\/jats:italic>\n                      1349C&amp;gt;T, and 94.7% sensitivity and 93.1% specificity for\n                      <jats:italic>katG<\/jats:italic>\n                      944G&amp;gt;C, using sequencing as the reference standard. Notably, the LFC-integrated assay maintained comparable diagnostic accuracy with a total turnaround time of 35\u00a0min (20\u00a0min for RPA, 5\u00a0min for CRISPR cleavage, and 10\u00a0min for lateral flow strip reading).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>The established multiplex-RPA CRISPR-AaCas12b assay enables simple, accurate, and sensitive detection of MTB and common mutations associated with MDR-TB. With a rapid, simplified workflow and low resource requirements, this approach holds considerable potential for point-of-care testing in resource-limited settings, thus facilitating improved surveillance and control of TB and drug-resistant TB.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fcimb.2026.1844184","type":"journal-article","created":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T05:39:30Z","timestamp":1779169170000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["Simultaneous molecular detection of Mycobacterium tuberculosis and multidrug resistance using CRISPR-AaCas12b-based nucleic acid assay"],"prefix":"10.3389","volume":"16","author":[{"given":"Yujiao","family":"Hu","sequence":"first","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dan","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanjun","family":"Diao","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Congxia","family":"Bai","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ke","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fang","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xi\u2019an Chest Hospital","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoyan","family":"Hao","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"liu","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiayun","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Medicine, Xijing Hospital, Fourth Military Medical University","place":["Xi\u2019an, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,5,19]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"1588","DOI":"10.1128\/jcm.00113-11","article-title":"Mutation detection and accurate diagnosis of extensively drug-resistant tuberculosis: report from a tertiary care center in India","volume":"49","author":"Ajbani","year":"2011","journal-title":"J. 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