基于单碱基延伸标签反应的常见食源性致病菌基因芯片检测方法的建立

Development of single base extension-tags microarray for the detection of food-borne pathogens

摘要:

针对8种常见的食源性致病菌(金黄色葡萄球菌、副溶血弧菌、单核细胞增生李斯特菌、沙门氏菌、阪崎肠杆菌、志贺氏菌、肠出血性大肠杆菌O157:H7和空肠弯曲杆菌), 建立了基于单碱基延伸标签反应原理的基因芯片检测方法。筛选和整合8种食源性致病菌基因组中的特异性序列和相应PCR引物, 致病菌靶DNA片段被扩增和纯化作为单碱基延伸标签反应的模板, 反应产物在DNA芯片上与探针进行杂交反应, 然后通过扫描基片对荧光强度结果进行判断。实验结果表明, 可采用基于单碱基延伸标签反应的基因芯片方法同时特异性检测8种食源性致病菌, 基因组DNA多重检测灵敏度可达到0.1 pg, 以鼠伤寒沙门氏菌为单一检测对象的细菌纯培养物灵敏度可达到5×102 CFU/mL。本方法可以快速灵敏地检测食源性致病菌, 为食源性疾病的诊断和防治提供了一个有效的方法。

Abstract:

We developed single base extension-tags (SBE-tags) microarray to detect eight common food-borne pathogens, including Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazaki, Shigella, Escherichia coli O157:H7 and Campylobacter jejuni. With specific PCR primers identified and integrated for eight food-borne pathogens, target sequences were amplified and purified as template DNA of single base extension-tags reaction. The products were hybridized to microarrays and scanned for fluorescence intensity. The experiment showed a specific and simultaneous detection of eight food-borne pathogens. The system limits is 0.1 pg for a genomic DNA and 5×102 CFU/mL for Salmonella typhimurium cultures. The single base extension-tags assay can be used to detect food-borne pathogens rapidly and accurately with a high sensitivity, and provide an efficient way for diagnosis and control of disease caused by food-borne pathogens.