摘要
[目的]利用可见光基因芯片技术,针对12种常见食源性致病菌,建立快速、准确、高通量的诊断方法。[方法]设计靶细菌16S rDNA和23S rDNA的通用引物,反向引物5’端标记生物素;特异寡核苷酸探针设计在两对引物之间的可变区,5’端标记氨基基团。将探针点样于固相载体制备基因芯片,优化PCR反应体系后,PCR产物与芯片点制探针区域进行杂交,然后通过化学显色直接观察结果,并评价反应体系的特异性、灵敏度、重复性等指标。收集临床样本28例,同时制备双盲模拟污染样本10例,对检测方法进一步评价。[结果]:本试验所建立的基因芯片方法可同时检测志贺菌、耶尔森氏菌、沙门菌、腊样芽孢杆菌、空肠弯曲菌、金黄色葡萄球菌、霍乱弧菌、副溶血性弧菌、单增李氏菌、布鲁氏菌、变形杆菌、肠出血性大肠杆菌O157:H7等12种食源性致病菌,特异性高,操作简便;针对纯培养食源性致病菌反应体系的灵敏度为103cfu/mL,模拟样本的灵敏度为104cfu/mL;重复性好;采用可见光基因芯片方法检测28例临床样本,26例与常规培养方法结果完全一致,符合率达到92.9%;双盲模拟样本的检测符合率达到100%。
It is to develop an accurate, rapid, and high - throughput assay based on visualized DNA microarray for detecting twelve foodborne pathogens. The 16S rDNA 和 23S rDNA genes were chosen simultaneously as targets for screening of specific probes and designing universal primers to detect and identify twelve foodborne pathogens. Biotin - labeled universal primers for amplification of bacterial 16S rDNA and 23S rDNA were used to amplify extracted genome DNA. The twelve foodborne pathogens were identified by hybridization of the PCR product with immobilized specific probes labeled with amino group on glass slide. Color development was achieved by adding POD and TMB. PCR reactions were optimized. Then the specificity, sensitivity and reproducibility were evaluated, and 28 clinical specimens and 10 double - blind samples were detected. The target pathogens detected by this assay described here were Listeria monocytogenes, Vibrio parahaemolyticus , Campylobacter jejuni, Staphylococcus aureus, Bacillus cereus, Salmonella spp. ,Shigella spp. ,Proteus spp. ,Brucella spp. ,Escherichia coli O157：H7, Yersinia spp. and Vibrio cholera. The assay was specific, rapid and simple. The minimum detection limit of DNA microarray was approximately 10^3cfu/mL. The limit for detecting mock samples such as food was 10^4cfu/mL. Among 28 clinical samples, 26 samples were positive for foodborne pathogens by the visualized DNA microarray method, which was the same to the results obtained from the conventional assays. The coincidence rate was 92.9% ; when 10 double - blind samples were detected by using visualized DNA microarray, the resuhs were in accordance with that of conventional methods. The coincidence was 100%.
出处
《检验检疫科学》
2009年第3期17-21,共5页
Inspection and Quarantine Science
关键词
可见光
基因芯片
食源性致病菌
检测
Visualized
DNA microarray
Foodborne pathogens
Detection
