Ultrasensitive and highly specific detection of the <i>Brucella</i> genus and <i>B</i> . <i>melitensis</i> by CRISPR/Cas12b‐multiple cross displacement amplification technique

Brucellosis is caused by members of the <i>Brucella</i> spp. and remains one of the world's major zoonotic diseases. <i>Brucella melitensis</i> (<i>B. melitensis</i>) as the most contagious <i>Brucella</i> species cannot be ignored as an essential source of infection for brucellosis, especially in countries/regions dominated by animal husbandry. Thus, the identification of the <i>Brucella</i> genus and <i>B. melitensis</i> is crucial for rapid diagnosis of brucellosis to control disease transmission and clinical treatment. Here, we developed the CRISPR/Cas12b nuclease combined with a multiple cross displacement amplification (MCDA) assay (CRISPR-MCDA) for highly specific and sensitive detection of <i>Brucella</i> genus and <i>B. melitensis</i> in clinical applications. Two sets of specific primers were designed targeting the specific gene of <i>Brucella</i> genus (<i>Bcsp31</i>) and <i>B. melitensis</i> (<i>BMEII0466</i>), respectively. The CRISPR-MCDA assay showed high specificity and sensitivity in 28 non-<i>Brucella</i> isolates and 64 clinical samples. The detection limit of CRISPR-MCDA assay was 2 copies/μL in the plasmid dilution template, and the whole detection process took within 90 minutes with nanoparticle-based lateral flow biosensor (LFB) to validate experimental results. Taken together, the CRISPR-MCDA-LFB assay is a visual, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for <i>Brucella</i> genus and <i>B. melitensis</i>.IMPORTANCEThe prevention and control of Brucellosis urgently require rapid and accurate diagnostic methods. This work validates a new method for the simultaneous detection of <i>Brucella</i> genus and <i>B. melitensis</i>. The method can effectively reduce the chances of contamination and provides a more rapid, sensitive, and specific on-site detection of <i>Brucella</i>. It also offers a solution for the rapid screening of Brucellosis in resource-limited environments, which is crucial for effective disease prevention and control. This technology can also be widely applied to the rapid detection of other pathogens beyond <i>Brucella</i>.