AUTHOR=Yang Xinggui , Wang Yue , Liu Ying , Huang Junfei , Wei Xiaoyu , Tan Qinqin , Zeng Xiaoyan , Ying Xia , Li Shijun 

TITLE=Rapid, ultrasensitive, and highly specific identification of Brucella abortus utilizing multiple cross displacement amplification combined with a gold nanoparticles-based lateral flow biosensor

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.1071928

DOI=10.3389/fmicb.2022.1071928

ISSN=1664-302X

ABSTRACT=<p><italic>Brucella abortus</italic> (<italic>B. abortus</italic>) as an important infectious agent of bovine brucellosis cannot be ignored, especially in countries/regions dominated by animal husbandry. Thus, the development of an ultrasensitive and highly specific identification technique is an ideal strategy to control the transmission of bovine brucellosis. In this report, a novel detection protocol, which utilizes multiple cross displacement amplification (MCDA) combined with a gold nanoparticles-based lateral flow biosensor (AuNPs-LFB) targeting the <italic>BruAb2_0168</italic> gene was successfully devised and established for the identification of <italic>B. abortus</italic> (termed <italic>B. abortus</italic>-MCDA-LFB). Ten specific primers containing engineered C1-FAM (carboxyfluorescein) and D1-biotin primers were designed according to the MCDA reaction mechanism. These genomic DNA extracted from various bacterial strains and whole blood samples were used to optimize and evaluate the <italic>B. abortus</italic>-MCDA-LFB assay. As a result, the optimal reaction conditions for the <italic>B. abortus</italic>-MCDA-LFB assay were 66°C for 40 min. The limit of detection of the <italic>B. abortus</italic>-MCDA-LFB was 10 fg/μl (~3 copies/μl) for genomic DNA extracted from pure cultures of <italic>B. abortus</italic> isolate. Meanwhile, the <italic>B. abortus</italic>-MCDA-LFB assay accurately identified all tested <italic>B. abortus</italic> strains, and there was no cross-reaction with non-<italic>B. abortus</italic> pathogens. Moreover, the detection workflow of the <italic>B. abortus</italic>-MCDA-LFB assay for whole blood samples can be completed within 70 min, and the cost of a single test is approximately 5.0 USD. Taken together, the <italic>B. abortus</italic>-MCDA-LFB assay is a visual, fast, ultrasensitive, low-cost, easy-to-operate, and highly specific detection method, which can be used as a rapid identification tool for <italic>B. abortus</italic> infections.</p>