水体中致病菌快速检测的基因芯片技术研究  

Studies on rapid detection of pathogens in water by gene chip technique

作　　者：王大勇[1] 方振东[1] 谢朝新[1] 敖漉[1] 肖进文 张春秀[3] 

机构地区：[1]解放军后勤工程学院营房管理与工程系,重庆401311 [2]重庆进出口食品安全检测技术工程中心,重庆400020 [3]上海生物芯片有限公司 生物芯片上海国家工程研究中心,上海201203

出　　处：《解放军医学杂志》2010年第9期1117-1122,共6页Medical Journal of Chinese People's Liberation Army

基　　金：重庆市科技攻关计划项目(CSTC2008AB7132)

摘　　要：目的探讨基因芯片技术行水体致病菌快速检测的方法。方法采用基于单碱基延伸标签反应的基因芯片方法,对金黄色葡萄球菌、肠炎沙门菌、单核细胞增生李斯特菌、志贺菌、军团菌、肺炎克雷伯菌、肠出血性大肠埃希菌O157:H7、幽门螺杆菌、大肠埃希菌、霍乱弧菌、结核分枝杆菌、鼠疫耶尔森菌、炭疽芽孢杆菌等13种可能存在于水体中的致病菌进行检测。结果该基因芯片可同时特异性地检测13种致病菌,基因组DNA检测灵敏度可达0.2pg,以肠炎沙门菌为单一检测对象的细菌纯培养物灵敏度可达1.0×103cfu/ml;配合浓缩集菌设备,整个检测时间可缩短至8h以内;用所制备的基因芯片检测模拟和实际水样,准确率达100%。结论该基因芯片检测水体致病菌操作简单,速度快,灵敏度高,稳定性和重复性好。Objective Contaminated water by pathogens can lead to outbreak and spread of various diseases in the public emergency such as terror attacks and natural disasters.In order to protect the army and people against infectious diseases which may occur in disasters,it is important to detect these pathogens accurately in time.In the present study,13kinds of pathogens possibly existed in water were taken as objects for detection,including Staphylococcus aureus,Salmonella enteritidis,Listeria monocytogenes,Shigella sp.,Legionella pneumophila,Klebsiella pneumoniae,EHEC O157：H7,Helicobacter pylori,Escherichia coli,Vibrio cholerae,Mycobacteriumt uberculosis,Yersinia pestis and Bacillus anthracis,and the rapid detection methods of the pathogens based on microarray gene chip technology was studied.Methods Fourteen pairs of specific primers were designed and divided into 3groups for multiplex PCR reactions：the first was Shi＋Sa＋O157＋Ec＋SEN,the second was Lm＋Lp＋Kp＋ Hp,and the third was MT＋BA＋YP ＋ VC＋16S.By combining the multiplex PCR with the gene chip technology,the SBE-tags primers were designed and Cy3fluorescent labeling reactions were performed,and the microarray gene chip detection method of 13pathogens in water was established.Combined with Milliflex Plus concentrator,a set of rapid detection method of pathogens in water based on microarray gene chip technology was established.Results The system limit was 0.2pg for a genomic DNA and 1.0×103cfu/ml for Salmonella cultures,respectively. Combined with Milliflex Plus concentrator,the treatment process of detection has been shortened to 8hours.The detective accuracy rate for simulated water samples was 100%.Conclusions The microarray gene chip detection method established in present study is simpler in operation,shorter in detection time,better in sensitivity,stability and repeatability compared with the traditional method.It laid a reliable theory and technology basis for detecting and identifying pathogens accurately,sensitively,with high throughput,

关 键 词：细菌 水 寡核苷酸序列分析 单碱基延伸标签反应 

分 类 号：R37-33[医药卫生—病原生物学]