Rapid and simultaneous analysis of five foodborne pathogenic bacteria using multiplex PCR
Rapid and simultaneous analysis of five foodborne pathogenic bacteria using multiplex PCR
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使用多重 PCR 快速同时分析五种食源性致病菌
DOI：
10.1007/s00217-013-2039-1
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发表时间：
2013-10-01
影响因子：
3.3
通讯作者：
Guan, Zheng Jun
中科院分区：
文献类型：
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作者：
Guan, Zheng Ping;Jiang, Yun;Guan, Zheng Jun
本研究的目的是开发一种使用纯细菌培养物和猪肉的快速多重 PCR (m-PCR) 方法，能够同时检测猪肉中可能存在的五种主要食源性病原体（金黄色葡萄球菌、单核细胞增生李斯特氏菌、大肠杆菌 O157:H7、沙门氏菌和小肠结肠炎耶尔森氏菌）。设计5对引物对沙门氏菌、L.单核细胞增多症，E.大肠杆菌O157：H7，S。金黄色葡萄球菌和Y。小肠结肠炎，分别。在确定 m-PCR 特异性的基础上，通过在培养基和猪肉中进行过夜富集步骤来执行敏感性。 16S rRNA 基因被作为细菌 DNA 存在的内部对照基因。结果表明，m-PCR 是同时检测五种目标病原体的有效方法，具有高特异性。过夜培养后，同时检测 5 种目标病原体的检出限为 103 cfu/mL，检测单一病原体的检出限低于 10 cfu/mL。 m-PCR 方案成功检测到在猪肉上一起接种过夜的所有五种微生物。沙门氏菌，S。金黄色葡萄球菌，L.单核细胞增多症，Y.小肠结肠炎和E.猪肉中检测到的大肠杆菌 O157:H7 水平分别为 142、51、9、33 和 670 cfu/mL。本研究开发的m-PCR检测可为猪肉安全的常规监测提供有效且信息丰富的补充。
The objective of this study was to develop a rapid multiplex PCR (m-PCR) method using pure bacterial cultures and pork that would allow the simultaneous detection of five major foodborne pathogens likely to be found in pork (Staphylococcus aureus,Listeria monocytogenes,Escherichia coliO157:H7,SalmonellaandYersinia enterocolitica). Five pairs of primers were designed to identifyinvAgene,hlyAgene,rfbEgene,nucgene andailgene forSalmonella,L. monocytogenes,E. coliO157:H7,S. aureusandY. enterocolitica, respectively. On the basis of determining specificity of the m-PCR, sensitivity was performed with an overnight enrichment step in culture media and pork. The 16S rRNA gene was targeted as an internal control gene of the presence of bacterial DNA. The results suggested that the m-PCR was an effective procedure having high specificity for the simultaneous detection of the five target pathogens. After overnight culture, the detection limit was 103cfu/mL for the simultaneous detection of the five target pathogens and less than 10 cfu/mL for detection of a single pathogen. The m-PCR protocol successfully detected all five organisms inoculated overnight together on pork.Salmonella,S. aureus,L. monocytogenes,Y. enterocolitcaandE. coliO157:H7 were detected at levels of 142, 51, 9, 33 and 670 cfu/mL, respectively, in the pork. The m-PCR assay developed in this study could provide an effective and informative supplement for routine monitoring for pork safety.