11种（株）食源性细菌基因芯片检测方法的建立.pdfVIP

生物技术通报 ·研究报告· BIOTECHNOLOGY BULLETIN 2013年第12期 11 种 （株 ）食源性细菌基因芯片检测方法的建立 高兴  辛文文  高姗  康琳  王景林 （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） 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。 关键词 ： 食源性细菌 基因芯片 多重PCR 特异性基因 检测 Development of a DNA Microarray for Detection of 11 - Food borne Pathogens Gao Xing Xin Wenwen Gao Shan Kang Lin Wang Jinglin （State Key Laboratory of Pathogen and Biosecurity ，Institute of Microbiology and Ep idemiology ，Academy of Military Medical Sciences ， Beij ing 10007 1 ） Abstract:  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