靶序列富集多重PCR结合DHPLC同时检测5种食源性致病菌

Target enriched multiplex PCR coupled with high-performance liquid chromatography assay for simultaneous detection of five foodborne pathogens

中文关键词:  靶序列富集多重PCR  变性高效液相色谱  沙门氏菌  单核细胞增生李斯特氏菌  大肠埃希氏菌O157  金黄色葡萄球菌  志贺氏菌

英文关键词:target enriched multiplex PCR  denaturing high-performance liquid chromatography  Salmonella  Listeria monocytogenes  Escherichia coli O157  Staphylococcus aureus  Shigella

中文摘要:

      目的 建立同时检测沙门氏菌、单核细胞增生李斯特氏菌、大肠埃希氏菌O157、金黄色葡萄球菌和志贺氏菌5种食源性致病菌的简便、灵敏的高通量方法。方法 根据GenBank公布的5种致病菌的特异性靶基因序列, 设计5对特异性引物, 与通用引物连接构成复合引物。经过反应条件的优化, 建立了5种致病菌的靶序列富集多重PCR (target enriched multiplex PCR, Tem-PCR)扩增方法, 扩增产物采用变性高效液相色谱(denaturing high-performance liquid chromatography, DHPLC)技术进行检测。结果 采用Tem-PCR-DHPLC方法, 检测48株试验菌株, 未发现非特异性结果。对5种致病菌检测灵敏度, 除金黄色葡萄球菌为620 cfu/mL外, 其余都小于100 cfu/mL。对4种样品的检测结果与国家标准方法相符合。结论 Tem-PCR-DHPLC检测方法, 相比传统多重PCR方法, 能有效地解决引物扩增效率不均衡的问题, 不需优化引物浓度, 方法特异性强, 灵敏度高, 具有较好的实用性。

英文摘要:

      [Objective] A novel target enriched multiplex PCR (Tem-PCR) coupled with high-performance liquid chromatography(DHPLC) assay（Tem-PCR-DHPLC） for the simultaneous detection of five foodborne pathogens: Salmonella, Listeria monocytogenes, Escherichia coli O157，Staphylococcus aureus and Shigella, was developed to deal with the uneven amplification in traditional multiplex PCR. [Methods] According to their specific target genes published in GenBank, 5 pairs of specific primers were designed and connected with the universal primer. The Tem-PCR-DHPLC assay was successfully established and assessed. [Results] Five foodborne pathogens were detected by Tem-PCR-DHPLC, and results showed it is specific, sensitive and practical. [Conclusion] Tem-PCR-DHPLC assay is a new method for high throughput detection.