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 denaturing high-performance liquid chromatography assay for simultaneous detection of 5 foodborne pathogens

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。

ABSTRACT:Objective To establish a convenient and sensitive method for the simultaneous detection of 5 foodborne pathogens:Salmonella, Listeria monocytogenes, Escherichia coli O157, Staphylococcus aureus and Shigella. Methods According to specific target genes published in GenBank, 5 pairs of specific primers connected with the universal primer were designed and termed the composite primers. Following optimization of reaction conditions, the target enriched multiplex PCR(Tem-PCR) assay for the detection of 5 foodborne pathogens was developed, and PCR products were separated by denaturing high performance liquid chromatography (DHPLC). Results Totally 48 bacterial strains were detected without nonspecific results. For the detection sensitivity of 5 foodborne pathogens, Staphylococcus aureus was 620 cfu/mL, the rest were less than 100 cfu/mL. The detection results of the samples were consistent with the national standard method. Conclusion Compared to conventional multiplex PCR assay, the Tem-PCR-DHPLC assay do not need to optimize concentration of each pairs of primers, which overcomes the uneven amplification in conventional multiplex PCR, and shows a high specificity and sensitivity, and with a good practicability.