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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-well microplate DNA diagnostic chip that can simultaneously detect nine common food-borne pathogens. Methods Staphylococcus aureus, Salmonella, Escherichia coli O157: H7 (Stx1 and Stx2), Shigella, Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Vibrio parahaemolyticus , Vibrio cholerae and other nine kinds of the most common food-borne pathogens, first select the appropriate conserved genes, the design of species-specific PCR primers (5’-end labeled biotin) and detection probe, Multiplex PCR amplification system was established. Then, the probe was spotted into the micropores of 96-well microplate in a 5 × 5 array format, and the microporous hybridization system of PCR products and immobilized probes was established by the optimization of conditions. Avidin alkaline phosphatase and the chemical chromogenic substrate NBT / BCIP to detect specific PCR hybridization products. Results Twenty strains of standard strains were used to verify the established DNA microarray DNA diagnostic chip for foodborne pathogens. The results obtained were quite specific and stable. In a food poisoning incident, the chip detected Staphylococcus aureus within 12 hours after sampling, consistent with the results of traditional bacterial isolation and culture and biochemical identification. Conclusion The new DNA microarray DNA diagnostic chip established in this study is rapid, accurate, automated and high-throughput. It provides a very valuable detection technique for rapidly coping with public health emergencies such as food poisoning .