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

() 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 gene was successfully devised and established for the identification of (termed -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 -MCDA-LFB assay. As a result, the optimal reaction conditions for the -MCDA-LFB assay were 66°C for 40 min. The limit of detection of the -MCDA-LFB was 10 fg/μl (~3 copies/μl) for genomic DNA extracted from pure cultures of isolate. Meanwhile, the -MCDA-LFB assay accurately identified all tested strains, and there was no cross-reaction with non- pathogens. Moreover, the detection workflow of the -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 -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 infections.