【作者】 高兴; 曲险峰; 孙伟; 李华宇; 辛文文; 高姗; 康琳; 王景林;
【Author】 GAO Xing1,QU Xian-feng2,SUN Wei2,LI Hua-yu2,XIN Wen-wen1,GAO Shan1,KANG Lin1,WANG Jing-lin1 1State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Beijing 100071,China 2Institute of Environment Protection and Inspection,No.63850 Troops,Baicheng,Jilin 137001,China
【机构】 军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室; 解放军63850部队防疫检验及环境监测所;
【摘要】 目的探讨随机PCR结合基因芯片技术用于多种食源性致病菌筛查检测的可行性。方法利用Clustal X和Oligo 6.0软件设计探针,进行生物信息学比对验证特异性,探针末端修饰后合成并制备基因芯片。使用锚定随机引物扩增细菌基因组DNA,偶联荧光染料的扩增产物与芯片杂交后扫描检测。对随机PCR及杂交反应等一系列检测条件进行优化。选取16种病原细菌进行芯片特异性验证,使用4种食源性致病菌DNA进行芯片灵敏度检测及重复性评价,制备细菌DNA混合样本和痢疾志贺菌模拟水污染样本对芯片检测效能进行初步考核。结果 9种食源性致病菌获得阳性结果,基因组DNA检测灵敏度102~103pg/μl,芯片重复性变异系数(CV)值<15%,混合样本检测与预期结果一致,最低可检测水样本中痢疾志贺菌的最低浓度为3.54×105cfu/ml。结论初步建立的基于随机引物PCR的基因芯片技术进行多种食源性致病菌检测的方法,为病原细菌高通量筛查检测提供了一种新的思路。 更多还原
【Abstract】 Objective To study the application of DNA microarray technique for screening and identifying multiple food-borne pathogens.Methods The oligonucleotide probes were designed by Clustal X and Oligo 6.0 at the conserved regions of specific genes of multiple food-borne pathogens,and then were validated by bioinformatic analyses.The 5’ end of each probe was modified by amino-group and 10 Poly-T,and the optimized probes were synthesized and spotted on aldehyde-coated slides.The bacteria DNA template in... 更多还原 【关键词】 食源性致病菌; 寡核苷酸序列分析; 随机扩增多态DNA技术; 【Key words】 food-borne pathogens; oligonucleotide array sequence analysis; random amplified polymorphic DNA technique; 【基金】 国家重大传染病防治科技重大专项(2008ZX10004-001)~~ 【分类号】R155.5
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