11种(株)食源性细菌基因芯片检测方法的建立  

Development of a DNA Microarray for Detection of 11 Food-borne Pathogens

作　　者：高兴[1] 辛文文[1] 高姗[1] 康琳[1] 王景林[1] 

机构地区：[1]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,北京100071

出　　处：《生物技术通报》2013年第12期123-128,共6页Biotechnology Bulletin

基　　金：国家重大传染病防治科技重大专项(2008ZX10004-001)

摘　　要：建立多重PCR反应结合基因芯片技术的11种(株)食源性致病菌检测方法。以金黄色葡萄球菌、志贺菌、霍乱弧菌、单增李斯特菌等11种(株)食源性细菌的特异性基因为靶基因,利用Premier 5.0和Oligo 6.0软件设计引物和探针,BLAST比对分析特异性。寡核苷酸探针点制醛基玻片制备基因芯片,13对引物分为3组扩增,PCR产物混合后与基因芯片杂交检测。鼠伤寒沙门菌、产气荚膜梭菌、奇异变形杆菌等11种(株)细菌使用该方法检测均能准确鉴定,基因组DNA灵敏度为10-100 pg,6组模拟DNA混合样本芯片检测结果与预期一致,18株沙门菌临床分离株检测均为阳性。从样本PCR扩增开始至检测完成,整个操作时间不超过3 h。该方法能够对11种(株)食源性致病菌快速准确鉴定,可以满足部分食源性致病菌通量检测的要求,具有较好的临床应用前景。It was to develop a microarray technique coupled with multiplex PCR for the detection and identification 11 food-borne pathogens. The specific gene of each pathogen such as Staphylococcus aureus , Shigellae , Vibrio cholerae and Listeria monocytogenes was chosen as the amplification target. The primers and probes were designed by Premier 5.0 and Oligo 6.0, and then were validated by BLAST. The oligonucleotide probes were immobilized on aldehyde-coated slides, and thirteen pairs of primers were divided into three groups. After the mixed PCR products had hybridization with DNA microarray, the hybridization images were acquired by ScanArray software. 11 food-borne bacteria such as Salmonella typhimurium , Clostridium perfringens and Proteus mirabilis were successfully identified by the array, and the detection limit of this assay was around 10--100 pg. The corresponding hybridization maps of six mixed bacteria DNA samples were obtained, and eighteen clinical isolates of Salmonella spp. were successfully tested. The testing process from PCR can be completed within 3 h. The results indicated that the method can be applied to identify 11 food-borne pathogens rapidly and accurately, and can meet the flux detection demand of multiple food-borne bacteria and has good prospect of clinical application.

关 键 词：食源性细菌 基因芯片 多重PCR 特异性基因 检测 

分 类 号：R155.5[医药卫生—营养与食品卫生学]