食品科学

• 分析检测 • 上一篇    下一篇

单增李斯特菌和金黄色葡萄球菌双重荧光定量PCR检测方法建立

邵美丽,董 鑫,赵燕丽,孔保华,刘思国   

  1. 1.东北农业大学食品学院,黑龙江 哈尔滨 150030;2.中国农业科学院哈尔滨兽医研究所,黑龙江 哈尔滨 150001
  • 出版日期:2013-08-25 发布日期:2013-09-03

A Duplex Fluorescence Quantitative PCR Assay for Detecting Listeria monocytogenes and Staphylococcus aureus

SHAO Mei-li,DONG Xin,ZHAO Yan-li,KONG Bao-hua,LIU Si-guo   

  1. 1. College of Food Science, Northeast Agricultural University, Harbin 150030, China;
    2. Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China
  • Online:2013-08-25 Published:2013-09-03

摘要:

根据GenBank公布的单增李斯特菌的hlyA基因序列和金黄色葡萄球菌的nuc基因序列各设计一对引物和一条探针,建立基于TaqMan探针的单增李斯特菌和金黄色葡萄球菌双重荧光定量聚合酶链式反应(PCR)检测方法。该方法对所有目标菌株均产生特异性扩增曲线,其他非目标菌均不产生扩增曲线,具有较强的特异性。且该法对阳性重组质粒pMD18-hlyA和pMD18-nuc同时定量扩增的敏感度分别为19.5拷贝/μL和18.7拷贝/μL。同步检测人工染菌肉样中单增李斯特菌和金黄色葡萄球菌的最低检出限均为102CFU/g。

关键词: TaqMan探针, 单增李斯特菌hlyA基因, 金黄色葡萄球菌nuc基因, 荧光定量聚合酶链式反应

Abstract:

A TaqMan duplex fluorescence quantitative PCR assay for detecting Listeria monocytogenes and
Staphylococcus aureus was presented. Specific primers and probes were designed according to the hlyA gene sequence of
Listeria monocytogenes and the nuc gene of Staphylococcus aureus published in GenBank. The developed assays detected
only the two pathogens, respectively, but not other bacteria, indicating the high specificity of the duplex PCR. When the
positive recombinant plasmids pMD18-hlyA and pMD18-nuc were simultaneously amplified, the sensitivity for them was
19.5 copy/μL and 18.7 copy/μL, respectively, and the limit of detection was 102 CFU/g for both Listeria monocytogenes and
Staphylococcus aureus in artificially inoculated meat.

Key words: TaqMan, hlyA gene of Listeria monocytogenes, nuc gene of Staphylococcus aureus, fluorescence quantitative polymerase chain reaction (PCR)