荧光定量PCR快速检测炭疽芽孢杆菌的实验研究

目的利用LightCycler建立一种简便、特异的实时荧光定量PCR检测方法,用于炭疽芽孢杆菌的快速检测。方法采用SYBR GreenⅠ随机掺入法,利用本实验室建立的实时荧光定量PCR反应体系,根据炭疽芽孢杆菌荚膜和水肿因子设计引物,同时检测两个毒力相关质粒的存在,检测其灵敏度和特异性,并以盲测试验进行验证;在此基础上鉴定14株炭疽芽孢杆菌,并测试该法检测土壤模拟污染标本的灵敏度,实验中设置内对照以排除假阴性结果的存在。结果炭疽杆菌基因组DNA的检测灵敏度可达每反应体系0.53pg,两个克隆株提取质粒的检测限分别为每反应体系12拷贝、140拷贝;检测14株炭疽芽孢杆菌及29株非炭疽芽孢杆菌的PCR扩增结果表明,炭疽芽孢菌DNA均出现特异的扩增结果,29株对照菌的DNA均为阴性;土壤模拟污染标本检测灵敏度可达每反应体系36个芽孢;20次重复实验结果表明其平均值与标准差为14.602±0.640。结论该方法检测炭疽芽孢杆菌具有简便、快捷、灵敏度高和特异性好的特点,为临床诊断、环境监测、卫生防疫等方面,快速检出炭疽芽孢杆菌提供了有利的手段。

Rapid detecoim of Bacillus anthracis with real-time PCR assays

To establish a simple and specific real-time PCR method for rapid detection of B.anthracis.The primers were designed based on the capsule and edema factor,two virulence-related plasmids could be detected simultaneously using real-time PCR reaction system with SYBR Green I established previously.The sensitivity and specificity assay were done and double blind experiment was used to validate its results.Then 14 B.anthracis strains were identified and the detection limits of spike soil samples were tested with presence of internal control.The sensitivity for genomic DNA detection was 0.53 pg per reaction,and for two clone-plasmids was 12 and 140 copies respectively.The detection results of 29 strains of non-B.anthracis and 14 B.anthracis strains showed the high specificity of this assay.For the spike samples,36 spores per PCR reaction system could be detected.It is concluded that the real-time PCR assay was simple and specific for rapid identification of B.anthracis,available for rapid diagnosis in case of emergency.