Brucellosis is a bacterial zoonotic disease that requires major attention for both health and financial facilities in many parts of the world including the Mediterranean and the Middle East. The existing gold standard diagnosis relies on the culturing technique, which is costly and time-consuming with a duration of up to 45 days. The Brucella protease biosensor represents a new detection approach that will lead to low-cost point-of-care devices for sensitive Brucella detection. In addition, the described diagnostic device is portable and simple to operate by a nurse or non-skilled clinician making it appropriate for the low-resource setting. In this study, we rely on the total extracellular protease proteolytic activity on specific peptide sequences identified using the FRET assay by high-throughput screening from the library of peptide (96 short peptides such as dipeptides and tripeptides) substrates for Brucella melitensis (B. melitensis). The B. melitensis-specific protease substrate was utilized in the development of the paper-based colorimetric assay. Two specific and highly active dipeptide substrates were identified (FITC-Ahx-K-r-K-Ahx-DABCYL and FITC-Ahx-R-r-K-Ahx-DABCYL). The peptide-magnetic bead nanoprobe sensors developed based on these substrates were able to detect the Brucella with LOD as low as 1.5 × 10² and 1.5 × 10³ CFU/mL using K-r dipeptide and R-r dipeptide substrates, respectively, as the recognition element. The samples were tested using this sensor in few minutes. Cross-reactivity studies confirmed that the other proteases extracted from closely related pathogens have no significant effect on the sensor. To the best of our knowledge, this assay is the first assay that can be used with low-cost, rapid, direct, and visual detection of B. melitensis.

中文翻译：

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基于荧光共振能量转移分析和纳米磁性颗粒的快速比色定量便携式平台，用于检测羊种布鲁氏菌

布鲁氏菌病是一种细菌性人畜共患疾病，需要世界许多地区（包括地中海和中东）的卫生和金融设施给予高度关注。现有的金标准诊断依赖于培养技术，该技术成本高昂且耗时，持续时间长达45天。布鲁氏菌蛋白酶生物传感器代表了一种新的检测方法，将导致用于灵敏布鲁氏菌检测的低成本护理点设备。此外，所描述的诊断装置是便携式的并且易于由护士或非熟练临床医生操作，使得其适合于资源匮乏的环境。在本研究中，我们依赖于使用 FRET 测定从肽库（96 种短肽，如二肽和三肽）底物中高通量筛选布鲁氏菌( B. melitensis ) 底物的特定肽序列上的总胞外蛋白酶蛋白水解活性。 ）。羊种芽胞杆菌特异性蛋白酶底物用于开发纸基比色测定法。鉴定出两种特异性且高活性的二肽底物（FITC-Ahx-KrK-Ahx-DABCYL 和 FITC-Ahx-RrK-Ahx-DABCYL）。基于这些基质开发的肽-磁珠纳米探针传感器能够分别使用Kr二肽和Rr二肽基质作为识别元件以低至1.5×10 ²和1.5×10 ^{3 CFU/mL的LOD检测}布鲁氏菌。使用该传感器在几分钟内测试了样品。交叉反应性研究证实，从密切相关的病原体中提取的其他蛋白酶对传感器没有显着影响。据我们所知，该测定是第一个可用于低成本、快速、直接和视觉检测羊种伯克霍尔德氏菌的测定。