{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T22:48:39Z","timestamp":1792622919321,"version":"4.5.0"},"reference-count":29,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,5,27]],"date-time":"2022-05-27T00:00:00Z","timestamp":1653609600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:p>Under the COVID-19 pandemic background, nucleic acid detection has become the gold standard to rapidly diagnose the infectious disease. A rapid, low cost, reliable nucleic acid detection platform will be the key to control next potential pandemic. In this study, a nucleic acid detection platform, which combined CRISPR\/Cas12a-based detection with loop-mediated isothermal amplification (LAMP), was developed and termed CRISPR-CLA. In the CRISPR-CLA system, LAMP preamplification was employed, and CRISPR\/Cas12a-based detection was used to monitor the preamplicons. The forward inner primer (FIP) was engineered with a protospacer adjacent motif (PAM) site TTTA of Cas12a effector at the linker region; thus, the CRISPR-CLA platform can detect any sequence as long as the primer design meets the requirement of LAMP. To demonstrate the validity of the CRISPR-CLA system, it was applied for the molecular diagnosis of nocardiosis caused by <jats:italic>Nocardia farcinica<\/jats:italic> (<jats:italic>N. farcinica<\/jats:italic>). A highly conserved and species-specific gene <jats:italic>pbr1<\/jats:italic> of <jats:italic>N. farcinica<\/jats:italic>, which was first reported in this study, was used as the target of detection. A set of LAMP primers targeting a fragment of <jats:italic>pbr1<\/jats:italic> of the <jats:italic>N. farcinica<\/jats:italic> reference strain IFM 10152 was designed according to the principle of CRISPR-CLA. Three CRISPR RNAs (crRNAs) with different lengths were designed, and the most efficient crRNA was screened out. Additionally, three single-strand DNA (ssDNA) probes were tested to further optimize the detection system. As a result, the <jats:italic>N. farcinica<\/jats:italic> CRISPR-CLA assay was established, and the whole detection process, including DNA extraction (20 min), LAMP preamplification (70\u00b0C, 40 min), and CRISPR\/Cas12a-mediated detection (37\u00b0C, 8 min), can be completed within 70 min. A fluorescence reader (for fluorescence CRISPR-CLA) or a lateral flow biosensor (for lateral-flow CRISPR-CLA) can be the media of the result readout. Up to 132 strains were used to examine the specificity of <jats:italic>N. farcinica<\/jats:italic> CRISPR-CLA assay, and no cross-reaction was observed with non-<jats:italic>N. farcinica<\/jats:italic> templates. The limit of detection (LoD) of the <jats:italic>N. farcinica<\/jats:italic> CRISPR-CLA assay was 100 fg double-strand DNA per reaction. <jats:italic>N. farcinica<\/jats:italic> was detected accurately in 41 sputum specimens using the <jats:italic>N. farcinica<\/jats:italic> CRISPR-CLA assay, which showed higher specificity than a real-time qPCR method. Hence, the <jats:italic>N. farcinica<\/jats:italic> CRISPR-CLA assay is a rapid, economic and accurate method to diagnose <jats:italic>N. farcinica<\/jats:italic> infection.<\/jats:p>","DOI":"10.3389\/fcimb.2022.884411","type":"journal-article","created":{"date-parts":[[2022,6,1]],"date-time":"2022-06-01T08:24:36Z","timestamp":1654071876000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":14,"title":["CRISPR\/Cas12a-Based Diagnostic Platform Accurately Detects Nocardia farcinica Targeting a Novel Species-Specific Gene"],"prefix":"10.3389","volume":"12","author":[{"given":"Xiaotong","family":"Qiu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuai","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xueping","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongtao","family":"Ren","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lichao","family":"Han","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenjun","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,5,27]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"1361","DOI":"10.1080\/22221751.2019.1664939","article-title":"CRISPR-Based Rapid and Ultra-Sensitive Diagnostic Test for Mycobacterium Tuberculosis","volume":"8","author":"Ai","year":"2019","journal-title":"Emerg. 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