Department of Microbiology, Guangzhou Center for Disease Control and Prevention, Guangzhou, China.
J Microbiol Methods. 2012 Sep;90(3):292-7. doi: 10.1016/j.mimet.2012.05.024. Epub 2012 Jun 9.
Brucella spp. are facultative intracellular bacteria that infect humans and animals. In this study, the loop-mediated isothermal amplification (LAMP) was used to detect the Brucella-specific gene omp25. Reaction conditions were optimized as temperature 65°C, reaction time 60 min, Mg(2+) concentration 8.0 mmol/L, polymerase content Bst DNA, 0.5 μL, deoxyribonucleotide concentration 1.6 mmol/L, and inner/outer primer ratio 1:8. The LAMP method was evaluated with 4 Brucella species and 29 non-Brucella bacteria species. Positive reactions were observed on all the 4 Brucella species but not on any non-Brucella species. The limit of detection of the LAMP method was 3.81 CFU Brucella spp. Using the LAMP method, 7 of 110 raw milk samples and 5 of 59 sheep blood samples were detected positive of Brucella spp. Results indicated that LAMP is a fast, specific, sensitive, inexpensive, and suitable method for diagnosis of Brucella spp. infection.
布鲁氏菌属是一种兼性细胞内细菌，可感染人类和动物。在本研究中，采用环介导等温扩增（LAMP）方法检测布鲁氏菌特异性基因 omp25。优化反应条件为温度 65°C，反应时间 60 min，Mg 2+浓度 8.0 mmol/L，聚合酶含量 Bst DNA 为 0.5 μL，脱氧核糖核苷酸浓度为 1.6 mmol/L，内/外引物比为 1:8。用 4 种布鲁氏菌和 29 种非布鲁氏菌细菌评价了 LAMP 方法。所有 4 种布鲁氏菌均呈阳性反应，但任何非布鲁氏菌均未呈阳性反应。LAMP 方法的检测限为 3.81 CFU 布鲁氏菌属。使用 LAMP 方法，在 110 份生乳样本中有 7 份和 59 份绵羊血液样本中有 5 份检测到布鲁氏菌属阳性。结果表明，LAMP 是一种快速、特异、敏感、廉价且适用于布鲁氏菌属感染诊断的方法。
