Laboratory of Diagnositics Development, China Animal Health and Epidemiology Center, 369 Nanjing Road, Qingdao, Shandong, 266032, China.
Laboratory of Diagnositics Development, China Animal Health and Epidemiology Center, 369 Nanjing Road, Qingdao, Shandong, 266032, China; College of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou, Jiangsu, 225009, China.
Mol Cell Probes. 2019 Dec;48:101451. doi: 10.1016/j.mcp.2019.101451. Epub 2019 Sep 18.
Brucella, the etiological agent of brucellosis, is an important zoonosis pathogen worldwide. Brucella infects humans and various domestic and wild animals, and represents a great threat to public health and animal husbandry. In the present study, we developed a real-time recombinase polymerase amplification (RPA) assay for the detection of Brucella. The assay targeted the bcsp31 gene of Brucella, and an RPA exo probe and a pair of primers were selected for assay validation. RPA sensitivity and specificity were evaluated using plasmid standards, Brucella representative strains, and non-Brucella strains. The RPA assay achieved a detection limit of 17 molecules in 95% of cases based on probit analysis, and could successfully distinguish 18 representative Brucella strains (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae and B. ovis), and four Brucella vaccine strains (A19, S19, S2 and M5). A total of 52 Brucella field strains were detected by real-time PCR and RPA in parallel, and compared with real-time PCR, the sensitivity of the RPA assay was 94% (49/52). Thus, this RPA assay may be a rapid, sensitive, and specific tool for the prevention and control of Brucellosis.
布鲁氏菌是布鲁氏菌病的病原体，是一种重要的人畜共患病病原体。布鲁氏菌感染人类和各种家养和野生动物，对公共卫生和畜牧业构成巨大威胁。在本研究中，我们开发了一种用于检测布鲁氏菌的实时重组酶聚合酶扩增（RPA）检测方法。该方法针对布鲁氏菌的 bcsp31 基因，选择 RPA 外切探针和一对引物进行检测验证。使用质粒标准品、布鲁氏菌代表株和非布鲁氏菌株评估 RPA 的灵敏度和特异性。基于概率单位分析，RPA 检测的灵敏度达到 95%时可检测到 17 个分子，可成功区分 18 个代表性布鲁氏菌株（B. abortus 生物型 1、2、3、4、5、6、7 和 9、B. melitensis 生物型 1、2 和 3、B. suis 生物型 1、2、3 和 4、B. canis、B. neotomae 和 B. ovis）和 4 株布鲁氏菌疫苗株（A19、S19、S2 和 M5）。通过实时 PCR 和 RPA 平行检测了 52 株布鲁氏菌田间分离株，与实时 PCR 相比，RPA 检测的灵敏度为 94%（49/52）。因此，该 RPA 检测方法可能是预防和控制布鲁氏菌病的一种快速、敏感和特异的工具。
