食源性致病菌的核酸可视微阵列检测技术  

Nucleic Acid Visual Microarray Detection of Food-Borne Pathogenic Bacteria

作　　者：李云霞[1] 马生龙[1] 聂莹莹[1] 马莉萍[1] LI Yunxia;MA Shenglong;NIE Yingying;MA Liping(Gansu Key Laboratory of Sensor and Sensing Technology,Institute of Sensor Technology,Gansu Academy of Sciences,Lanzhou 730000,China)

机构地区：[1]甘肃省科学院传感技术研究所甘肃省传感器与传感技术重点实验室,甘肃兰州730000

出　　处：《食品与生物技术学报》2020年第9期105-111,共7页Journal of Food Science and Biotechnology

基　　金：甘肃省科学院应用开发项目(2016JK-07);甘肃省科技计划资助项目(18JR3RA249)。

摘　　要：建立了一种快速检测3种食源性致病菌的核酸可视微阵列。分别设计与沙门氏菌invA基因、金黄色葡萄球菌nuc基因、志贺氏菌ipaH基因两端互补的基片探针和检测探针,设计3种基因的特异靶序列,特异靶序列或基因组DNA一端与固定在玻片上的氨基化基片探针杂交,另一端与巯基化检测探针连接形成复合体。使胶体金与检测探针结合,通过银染放大信号,检测3种食源性致病菌DNA。结果表明:通过对检测探针分别与基因组DNA、PCR产物及特异靶序列的杂交银染结果比较,3种结果都显示阳性,表明该方法可直接利用基因组DNA实现对食源性微生物的快速、高效检测,对3种食源性致病菌DNA的检测下限为1 pmol/L,在1 mmol/L^1 pmol/L浓度范围内得到肉眼可见的清晰结果。探针与基因组DNA的杂交实验结果表明,微阵列对3种食源性微生物的检测具有较好的特异性。说明该技术可快速、简便检测食源性微生物,市场前景广阔。A visualized microarray for rapid detection of nucleic acid of three food-borne pathogenic bacteria was developed.Substrate and detection probes which were complementary to both ends of Salmonella invA,Staphylococcus aureus nuc and Shigella ipaH genes were designed,respectively.Specific target sequences of three genes were also designed.One end of the specific target sequence or genomic DNA was hybridized with the amination probe fixed on the slide,and the other end was complementary to the sulfhydrylation probe to form a complex.The detection probe was connected with colloidal gold,and the signal was amplified by silver staining for the detection of food-borne pathogenic bacteria.Through comparative analysis of hybridization silver staining with genomic DNA,PCR products and specific target sequences,all results were positive,which demonstrated that the method could directly use genomic DNA to detect food-borne microorganisms rapidly and efficiently.The detection limit of genomic DNA of three foodborne pathogenic bacteria was 1 pmol/L,and clear results could be observed by naked eyes in the concentration range of 1 mmol/L^1 pmol/L.The results of hybridization between probe and genomic DNA indicated that visual microarray had good specificity for the detection of microorganisms.Therefore,this method can detect food-borne microorganisms quickly and easily,and has broad market prospects.

关 键 词：食源性致病菌 基因组DNA 可视化微阵列 银染 

分 类 号：R446.5[医药卫生—诊断学]