Mo Qiu-Hua, Li Qiang, Lin Ji-Can, Tan Hua, Tu Cheng-ning, Ye Li-qing, Liu Zhi-Ming, Du Jian, Sun Hong, Li Shu-xiang, Wang Sino, Yang Ze
Zhuhai Entry-Exit Inspection and Quarantine Bureau, Zhuhai, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2010 Mar;30(3):417-21.
To develop a 96-microwell plate DNA diagnostic chip for simultaneous detection of 9 major foodborne bacteria.
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 5x5 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.
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.
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.
研制一种用于同时检测9种主要食源性病原体的96孔板DNA诊断芯片。
根据金黄色葡萄球菌、沙门氏菌属、大肠埃希菌O157:H7（stx1和stx2）、志贺氏菌属、单核细胞增生李斯特菌、蜡样芽孢杆菌、小肠结肠炎耶尔森氏菌、霍乱弧菌和副溶血性弧菌这9种主要食源性病原体的保守基因，设计生物素标记的型特异性PCR引物和寡核苷酸探针。建立了一种用于同时扩增这些病原体的单管多重PCR体系，并将DNA探针以5×5阵列形式点样并固定在微孔板的孔中。经过条件优化后，在微孔中建立了PCR产物与寡核苷酸探针之间稳定的杂交体系。采用碱性磷酸酶标记的链霉亲和素和NBT/BCIP检测杂交后的PCR产物。
用20株标准菌株对96孔板DNA诊断芯片进行验证，获得了高度特异且稳定的实验结果。利用该芯片检测方法，在一次食物中毒事件采样后12小时内鉴定出致病病原体金黄色葡萄球菌，结果与传统细菌培养和生化鉴定结果一致。
新型96孔板DNA诊断芯片可实现快速、准确、自动化和高通量的细菌检测，对食物中毒等突发公共卫生事件的快速响应具有重要价值。
