Feng Na, Zhou Yazhou, Fan Yanxiao, Bi Yujing, Yang Ruifu, Zhou Yusen, Wang Xiaoyi
Anhui Medical University, Anhui, People's Republic of China; Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and Biosecurity, Laboratory of Analytical Microbiology, Beijing, China.
Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and Biosecurity, Laboratory of Analytical Microbiology, Beijing, China.
Braz J Microbiol. 2018 Jan-Mar;49(1):128-137. doi: 10.1016/j.bjm.2017.03.014. Epub 2017 Aug 26.
We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23CFU for pure culture, whereas 2.3×10 or 2.3×10CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3×10CFU, but PCR was negative at the level of 2.3×10CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3×10 or 2.3×10CFU, whereas 2.3×10 or 2.3×10CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.
我们开发了一种环介导等温扩增（LAMP）检测方法，通过靶向染色体上的3a序列来检测鼠疫耶尔森菌。使用耶尔森菌属的所有11个物种来评估LAMP和PCR的特异性，结果表明引物具有高度特异性。对于纯培养物，LAMP或PCR的灵敏度分别为2.3或23CFU，而对于模拟脾脏和肺脏样本，灵敏度分别为2.3×10或2.3×10CFU。对于模拟肝脏样本，LAMP的灵敏度为2.3×10CFU，但PCR在2.3×10CFU水平呈阴性。模拟脾脏和肺脏样本经磁珠处理后，LAMP或PCR的灵敏度为2.3×10或2.3×10CFU，而磁珠处理的肝脏样本灵敏度为2.3×10或2.3×10CFU。这些结果表明组织中的某些成分可抑制LAMP和PCR，且肝脏组织样本对LAMP和PCR的抑制作用强于脾脏和肺脏组织样本。LAMP比PCR具有更高的灵敏度，磁珠捕获DNA可显著提高LAMP的灵敏度。LAMP是一种简单、快速且灵敏的检测方法，适用于现场或贫困地区应用。
