论文

空肠弯曲菌环介导等温扩增检测方法的建立和优化

  • 许紫建 ,
  • 杨兵 ,
  • 苏霞 ,
  • 周宏专 ,
  • 史爱华 ,
  • 徐福洲
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  • 北京市农林科学院畜牧兽医研究所畜禽疫病防控技术北京市重点实验室, 北京 100097
许紫建(1982-),男,山东青岛人,在读硕士,主要从事动物病原细菌学研究

收稿日期: 2013-03-05

  修回日期: 2013-03-05

  网络出版日期: 2014-10-14

基金资助

北京市优秀人才项目(2011D002020000004);2011年度北京市留学人员科技活动择优资助项目;北京市农林科学院青年科学基金项目

Development and Optimization of a Loop-mediated Isothermal Amplification Assay for Detection of Campylobacter jejuni

  • Xu Zijian ,
  • Yang Bing ,
  • Su Xia ,
  • Zhou Hongzhuan ,
  • Shi Aihua ,
  • Xu Fuzhou
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  • Beijing Key Laboratory for Prevention and Control of Infectious Diseases in Livestock and Poultry, Institute of Animal Husbandry and Veterinary Medicine,Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097,China

Received date: 2013-03-05

  Revised date: 2013-03-05

  Online published: 2014-10-14

摘要

首先根据空肠弯曲菌mapA基因序列设计LAMP引物,建立检测空肠弯曲菌的LAMP检测方法。继而对LAMP方法中不同反应组分的浓度分别进行筛选,结果显示,当内外引物浓度分别为1.6,0.2μmol/L、Mg2+。浓度为8mmol/L、dNTP浓度为1.4 mmol/L、Betaine浓度为0.8 mol/L时获得最佳的反应结果。最后对LAMP反应的特异性和敏感性进行检测,特异性检测结果显示,对其他13种革兰氏阴性和革兰氏阳性细菌扩增结果均为阴性;敏感性检测结果显示,对空肠弯曲菌基因组DNA的检测限为每个反应管100 fg,对空肠弯曲菌培养菌液检测限为每个反应管7.5cfu。试验建立的LAMP方法为快速简便地自动物源性产品中检测空肠弯曲菌奠定了基础。

本文引用格式

许紫建 , 杨兵 , 苏霞 , 周宏专 , 史爱华 , 徐福洲 . 空肠弯曲菌环介导等温扩增检测方法的建立和优化[J]. 华北农学报, 2013 , 28(3) : 222 -226 . DOI: 10.3969/j.issn.1000-7091.2013.03.040

Abstract

In this study, the specific mapA gene was used to design a set of primers to develop a loop-mediated isothermal amplification ( LAMP) assay for detection of C. jejuni. To optimize the LAMP assay, the reaction components in the assay were screened to determine the optimal concentration. The final LAMP assay comprised 1.6μmol /L each of inner primers FIP and BIP,0.2 μmol /L each of outer primers F3 and B3,8 mmol /L Mg2 + ,1.4mmol /L dNTP,0 .8 mol /L Betaine. The specificity test showed that the assay correctly identified all C. jejuni strains but not 13 other bacterial species. The sensitivity of the assay was 100 fg per test tube for C. jejuni genomic DNA and 7. 5 cfu per test tube for C. jejuni bacterial culture. This LAMP assay is a rapid and simple tool for detection of C. jejuni and will provide an essential role in facilitating early diagnosis of this organism from animal products.

参考文献

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