食源致病菌96微孔板DNA诊断芯片的研制及在一起食物中毒突发事件中的应用  

Preparation of a 96-microwell plate DNA diagnostic chip for detection of foodborne bacteria and its application in an incident of food poisoning

作　　者：莫秋华[1] 李强[2] 林继灿[1] 谭华[1] 涂承宁[1] 叶立青[1] 刘志明[1] 杜坚[1] 孙虹[1] 李书香[1] 王献煌 杨泽[1] 

机构地区：[1]珠海出入境检验检疫局,广东珠海519020 [2]南方医科大学南方医院检验科,广东广州510515 [3]晶宇生物科技实业股份有限公司

出　　处：《南方医科大学学报》2010年第3期417-421,共5页Journal of Southern Medical University

基　　金：国家质检总局科技计划项目(2009IK206)

摘　　要：目的研制能同时检测9种常见食源致病菌的96微孔板DNA诊断芯片。方法针对金黄色葡萄球菌、沙门氏菌、大肠杆菌O157:H7(Stx1和Stx2)、志贺氏菌、产单核细胞李斯特菌、蜡样芽胞杆菌、小肠结肠炎耶尔森菌、副溶血弧菌、霍乱弧菌等9种最常见的食源性致病菌,首先选择合适的保守基因,设计种特异性的PCR引物(5'端标记生物素)和检测探针,通过参数优化建立一管多重PCR扩增体系;然后按5×5的阵列格式将探针点制到96微孔板的微孔内,通过条件优化建立稳定的PCR产物与固化探针的微孔杂交体系;采用链霉抗生物素蛋白碱性磷酸酶和化学显色底物NBT/BCIP来检测特异性的PCR杂交产物。结果20株标准菌株验证已建立的食源致病菌微孔板DNA诊断芯片,获得比较特异和稳定的实验结果。在一起食物中毒事件中,该芯片在采样后12h内检出金黄色葡萄球菌,与传统的细菌分离培养和生化鉴定的结果相符。结论本研究建立的新型食源致病菌96微孔板DNA诊断芯片具有快速、准确、自动化和高通量等特点,为快速应对食物中毒等突发公共卫生事件提供了非常有价值的检测技术。Objective To develop a 96-microwell plate DNA diagnostic chip for simultaneous detection of 9 major foodborne bacteria. Methods Type-specific PCR primers labeled with biotin and oligonucleotide probes were designed according to the conservative genes of 9 major foodborne bacteria Staphylococcus aureus, Salmonella spp., Escherichia coli O157：H7 （Stx1 and Stx2）, Shigella spp., Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Vibrio cholerae and Vibrio parahaemolyticus. A one-tube multiplex PCR system for simultaneous amplification of these bacteria was established, and the DNA probes were spotted and immobilized in the wells of the plate in 5×5 array format. Stable hybridization system between PCR products and oligonucleotide probes in the microwell was established after condition optimization. Alkaline phosphatase-conjugated streptavidin and NBT/BCIP were used to detect the hybridized PCR products. Results Twenty standard bacteria strains were used to validate the 96 microwell plate DNA diagnostic chip and highly specific and stable experiment results were obtained. Using this chip assay, the causal pathogen Staphylococcus aureus was identified within 12 h after the sampling from an incident of food poisoning, and the result was consistent with that obtained using conventional bacterial culture and biochemical identification. Conclusion The novel 96 microwell plate DNA diagnostic chip allows rapid, accurate, automated and high-throughput bacterial detection and is especially valuable for quick response to such public health emergencies as food poisoning.

关 键 词：食源致病菌 96微孔板芯片 DNA检测 食物中毒 

分 类 号：R595.7[医药卫生—内科学]