通用型基因悬浮芯片检测生物恐怖细菌的方法研究  

Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria

作　　者：文海燕[1] 王静[1] 刘衡川[2] 孙肖红[1] 杨宇[1] 胡孔新[1] 单麟军[1] 

机构地区：[1]中国检验检疫科学研究院卫生检疫研究所,北京100025 [2]四川大学华西公共卫生学院

出　　处：《中华预防医学杂志》2009年第10期890-894,共5页Chinese Journal of Preventive Medicine

基　　金：“十一五”国家科技支撑计划（2006BAK10B07）;质检公益性行业科研专项（2007GYJ023） 志谢 感谢军事医学科学院微生物流行病研究所杨瑞馥研究员、端青研究员、宋亚军博士、翟俊辉博士、郭兆彪老师在实验上的指导及支持

摘　　要：目的建立快速、高通量的基因悬浮芯片检测方法，用于生物恐怖病原体的快速筛检。方法选择保守的细菌16S rDNA序列，并针对炭疽芽孢杆菌（Bacillus anthracis，B．a）、鼠疫耶尔森菌（Yersinia pestis，Y.p）、布鲁菌属（Brucella spp.，Bru）、土拉弗朗西斯菌（Francisella tularensis，F.t）和类鼻疽伯克霍尔德菌（Burkholderia pseudomallei，B．P）等5种生物恐怖细菌设计种（属）特异性探针，经16S rDNA通用引物341A、519B扩增基因组DNA，用基因悬浮芯片方法进行检测，并对方法的灵敏度、特异度、重复性、检测能力进行验证。结果经16S rDNA通用引物扩增，特异性探针杂交检测，能将样本细菌鉴定到属水平，同属菌出现交叉反应；检出限分别为类鼻疽伯克霍尔德菌1．5pg／μl，布鲁菌属20pg／μl，炭疽芽孢杆菌7pg／μl，土拉弗朗西斯菌0．1pg／μl，鼠疫耶尔森菌1．1pg／μl；15次重复检测各探针变异系数（CV）为5．18％～17．88％，具有较好的重复性；方法可正确检出模拟白色粉末样本中炭疽芽孢杆菌和鼠疫耶尔森菌。结论建立了通用型基因悬浮芯片检测体系快速高通量筛查生物恐怖细菌的方法。Objective To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biotbreat bacteria. Methods 16 S rDNA universal primers for Bacillus anthracis , Francisella tularensis , Yersinia pestis , Brucella spp. and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B,the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. Results After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/μl （Burkholderia pseudomallei） ,20 pg/μl （ Brucella spp. ）,7 pg/μl （Bacillus anthracis ） , 0. 1 pg/ μl （ Francisella tulareusis ） , and 1.1 pg/μl （Yersinia pestis） ,respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17. 88% ,it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. Condusion The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

关 键 词：DNA引物 电泳 微芯片 生物恐怖 细菌 

分 类 号：R185[医药卫生—流行病学]