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ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Veterinary and Zoonotic Infection
Volume 15 - 2025 | doi: 10.3389/fcimb.2025.1545953
A CRISPR/cas13a-assisted precise and portable test for Brucella nucleic acid detection
Provisionally accepted
Haiwen Liu 1,2
Ling Xu 3
Ying Xiu 4 Na Ta 5
Qingqing Xu 1 Yu Fan 1
Kun Li 1 Hongyan Zhao 1
Dongri Piao 1
Feng Ren 3*
Hai Jiang 1*- 1 National Institute for Communicable Disease Control and Prevention (China CDC), Beijing, Beijing Municipality, China
- 2 Ulanqab Center for Disease Control and Prevention, Jining, China
- 3 Institute of Liver Diseases, Beijing Youan Hospital, Capital Medical University, Beijing, China
- 4 Inner Mongolia Autonomous Region Animal Disease Control Center, Hohhot, China
- 5 Inner Mongolia Center for Disease Control and Prevention, Huhhot, Inner Mongolia Autonomous Region, China
Brucella infection in humans or animals can lead to brucellosis, which has the potential to significantly impact both the economy and public health. Currently, molecular biological methods for diagnosing brucellosis are either complex or have low sensitivity, and it is difficult to apply them in real-life settings in the field. Therefore, this study aims to establish a rapid and convenient nucleic acid-based molecular biology method for on-site rapid detection of Brucella and early clinical screening of brucellosis. Based on the conserved sequence of the Brucella Bcsp31 gene, we designed CRISPR RNA (crRNA) and RAA primers. We developed a fluorescence detection method and a paper strip detection method by integrating RAA amplification with CRISPR/Cas13a detection. We applied these methods to analyze 100 samples of suspected brucellosis-infected milk, 123 samples of human whole blood, and 100 samples of sheep vaginal swabs in order to validate their practical utility. The RAA-CRISPR/Cas13a Brucella fluorescence detection method and the strip test method had detection limits of 10 0 copies/μL and 10 1 copies/μL, respectively, and both methods had a specificity of 100%. The positivity rate of the RAA-CRISPR/Cas13a fluorescence detection method for the milk, human whole blood, and sheep vaginal swab samples was 93% (93/100), 82.12% (101/123), and 91% (91/100), respectively; the strip test method, 87% (87/100), 64.23% (79/123), and 76% (76/100), respectively. In this study, we have developed a RAA-CRISPR detection method based on the Brucella BCSP31 gene, with potential applications in the identification of Brucella nucleic acid and implications for clinical diagnosis of brucellosis.
Keywords: Brucella1, Brucellosis2, CRISPR-Cas13a3, Fluorescence detection4, Nucleic acid detection5, RAA6, Strip test7, zoonosis8
Received: 16 Dec 2024; Accepted: 05 Mar 2025.
Copyright: © 2025 Liu, Xu, Xiu, Ta, Xu, Fan, Li, Zhao, Piao, Ren and Jiang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Feng Ren, Institute of Liver Diseases, Beijing Youan Hospital, Capital Medical University, Beijing, 100069, China
Hai Jiang, National Institute for Communicable Disease Control and Prevention (China CDC), Beijing, Beijing Municipality, China
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.