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沙门菌实时荧光定量PCR检测试剂盒的研制与应用
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孔繁德1，2，彭海滨3，徐淑菲2，陈琼4，陆承平1
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（1.南京农业大学 动物医学院，江苏 南京210095； 2.厦门出入境检验检疫局，福建 厦门361012；
3.福建农林大学 动物科学学院，福建 福州351006；
4.厦门市农产品质量安全检验测试中心，福建 厦门361009）
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摘要：
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根据沙门菌保守的fimY基因序列设计合成了引物和TaqMan探针，建立了快速检测沙门菌的实时荧光定量方法，并对反应条件进行了优化，组装成快速检测试剂盒。通过对临床样品的检测，该试剂盒的检测灵敏度达4.5CFU(25μL反应体系)，比常规PCR高100倍，而且稳定性良好，至少可以冷冻保存9个月。结果表明，所建立的沙门菌实时荧光定量PCR检测试剂盒具有操作简便、快速、特异性强、灵敏度高、重复性好、稳定性强等优点，适于大量样品的检测。 |
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关键词：
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沙门菌；TaqMan探针；实时荧光定量PCR；检测 |
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中图分类号：S 852.612 文献标识码：
A 文章编号：1673-4696(2006)08-0625-05 |
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Development and application of the real-time fluorescence
quantitative PCR detection kit for Salmonella |
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KONG Fan-de1,2,PENG Hai-bin3，XU
Shu-fei2，CHEN Qiong4，LU Cheng-ping1 |
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(1.College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;
2.Xiamen Entry-Exit Inspection and Quarantine Bureau，Xiamen 361012,China;
3.College of Animal Science,Fujian Agricultural and Forestry University，Fuzhou 351006,China;
4.Xiamen Agricultural Product Quality and Safety Testing Center，Xiamen 361009,China) |
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Abstract: The Taqman probe and primers were designed and synthesized according to the conserved gene fimY of Salmonella available in GenBank,and then reaction parameters were optimized to develop a real time Taqman quantitative PCR detection kit.The developed quantitative PCR kit could detect 4.5CFU in the 25μL-reaction-system.Its sensitivity was 100 times higher than that of the routine PCR,and it could be stored over nine months at -20℃.It was confirmed through detection of some clinic samples that the developed kit was the simple,sensitive,specific,accurate,good repeated and stable method for the rapid detection of Salmonella and could be applied to detection of a large number of samples. |
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Key words: Salmonella; TaqMan probe; real-time fluorescence quantitative PCR; detection |
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