Ultrasensitive and highly specific detection of the genus and . by CRISPR/Cas12b-multiple cross displacement amplification technique.

Brucellosis is caused by members of the spp. and remains one of the world's major zoonotic diseases. () as the most contagious 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 genus and 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 genus and in clinical applications. Two sets of specific primers were designed targeting the specific gene of genus () and (), respectively. The CRISPR-MCDA assay showed high specificity and sensitivity in 28 non- 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 genus and .IMPORTANCEThe prevention and control of Brucellosis urgently require rapid and accurate diagnostic methods. This work validates a new method for the simultaneous detection of genus and . The method can effectively reduce the chances of contamination and provides a more rapid, sensitive, and specific on-site detection of . 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 .