期刊文献+

建立环介导等温扩增方法检测腹泻患者中小肠结肠炎耶尔森菌 被引量:3

Development of a loop-mediated isothermal amplification for detection of Yersinia enterocolitica in patients with diarrhea
收藏 分享 导出
摘要 目的建立快速的检测方法对腹泻患者中的小肠结肠炎耶尔森菌进行快速检测。方法利用析因设计试验方法建立并优化检测小肠结肠炎耶尔森菌的环介导等温扩增技术(LAMP),并与普通PCR法对临床腹泻标本进行检测比较。结果析因设计试验确定LAMP最佳反应体系:Mg2++为2.0mmol,内引物FIP/BIP为1.6umol,dNTPs为1.6mmol,时间为60rain,反应温度为63℃。对12种细菌进行LAMP扩增,仅小肠结肠炎耶尔森菌获得阳性扩增结果,小肠结肠炎耶尔森菌基因组DNA和纯培养物的检测灵敏度分别为38.05fg和15CFU/ml。对腹泻样本进行直接检测,检测限为150CFU/g。539例腹泻患者粪便标本中,LAMP成功检出13例样本中含有小肠结肠炎耶尔森菌,普通PCR检出11例,灵敏度为100%,特异度为99.62%。结论本方法检测小肠结肠炎耶尔森菌特异性强、灵敏度高.且操作简单、快速、检测成本低.适合基层单位及现场廊用. Objective To develop a rapid method for detection Yersinia enterocolitica in patients with diarrhea. Methods Loop-mediated isothermal amplification (LAMP) reaction condition was optimized based on factorial experimental design. PCR and the developed LAMP methods were applied in clinical faecal samples and compared to evaluate its application. Results The optimal LAMP condition was containing 1.6 p~mol/L inner primer (FIP and BIP), 1.6 mmol/L dNTPs, 2.0 mmol/L MgC12 in 25 p~l volumes, which was confirmed by factorial experiment analysis. And the amplification program was as follows: 63~C for 60 rain and then heated at 80~C for 10 min. Only genomic DNA from Yersinia enterocolitica strains was positively detected by LAMP, while no LAMP products were obtained when detecting non- Yersinia strains. LAMP method could detect Yersinia enterocolitica genomic DNA as low as 38.05 fg, while for the pure culture bacteria was 15 CFU/ml. In artificially infected faecal specimens, the detection limit was 150 CFU/g. With regard to the 539 clinical specimens from diarrhea patients, 13 were tested positive using the LAMP assay, and 11 were tested positive using the PCR assay, resulted in the sensitivity of 100% and specificity of 99.62%. Conclusions The optimized LAMP method was proved to be sensitive and specific, easy-to-use and low cost. LAMP is readily to applied in primary care clinics.
作者 罗敏红 胡婷婷 冀新凤 李美青 黄燕惠 陈慧芳 姚月娴 陈清 俞守义 LUO Min-hong1, HU Ting-ting1, JI Xin-feng2, LI Mei-qing1, HUANG Yan-huP, CHEN Hui-fang~, YAO Yue-xian1, CHEN Qingl, YU Shou-yP (1.Southern Medical University, School of Public Health and Tropical Medicine, Guangdong , Guangzhou 510515 ; 2. First Affiliated Hospital of Shantou University, Guangdong , Shantou 515041, China)
出处 《热带医学杂志》 CAS 2013年第4期449-452,495共5页 Journal Of Tropical Medicine
关键词 小肠结肠炎耶尔森菌 析因设计 环介导等温扩增技术(LAMP) 腹泻 Yersinia enterocolitica factorial experiment design loop-mediated isothermal amplification (LAMP) diarrhea
作者简介 罗敏红(1986-),女,在读研究生,研究方向为分子流行病 通讯作者:俞守义(1937-),男,教授,博士生导师,研究方向为分子流行病.E-mail:qingchen@fimmu.com
  • 相关文献

参考文献10

二级参考文献55

  • 1杜之渝,吴宁玲,张大勇,郭红,郑晴,晏丕松.准分子激光原位角膜磨镶术后角膜基质床厚度安全值分析[J].中华眼科杂志,2004,40(11):741-744. 被引量:46
  • 2杨成 ,李滨 ,刘同慎 ,赵冬梅 ,胡凤爱 .电针对脊髓损伤后星形胶质细胞增生的影响[J].中国针灸,2005,25(8):569-572. 被引量:16
  • 3欧阳伟 HuangYL DaCD.电针减少大鼠神经元缺血性损伤并增强碱性成纤维生长因子的表达[J].山西医科大学学报,2000,31:67-8. 被引量:3
  • 4贺石林.医学科研办法导论[M].北京:人民卫生出版社,1998:201-207. 被引量:1
  • 5Stoner L, Sabatier MJ, Mahoney ET, et al. Electrical stimulation evoked resistance exercise therapy improves arterial health after chronic spinal cord injury [J]. Spinal Cord, 2007, 45(1): 49--56. 被引量:1
  • 6Saleh M, Soliman H, EI-Matbouli M. Loop-mediated isother real amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish [J]. BMCVet Res, 2008, 8 (12):1746-1756. 被引量:1
  • 7Notorni T, Okayama H, Masubuchi H, et al . Loop-mediated isothermal amplification of DNA [J] .Nucleic Acids Res, 2000, 28 (12):63. 被引量:1
  • 8Endo S, Komofi T, Ricci G, etal. Detection of S043 of Para coccidioides brasiliensis by the loop-mediated isotherm alamplification (LAMP) method [J]. FEMS Microbiol Lett, 2004 234 (1):93-97. 被引量:1
  • 9Okafuji T, Yoshida N, Fujino M, et al. Rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification [J]. J Clin Microbiol, 2005, 43 (4): 1625- 1631. 被引量:1
  • 10lhira M, Yoshikawa T, Enomoto Y, et al. Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification [J]. J Clin Microbiol, 2004, 42 (1):140-145. 被引量:1

共引文献28

同被引文献72

引证文献3

二级引证文献8

投稿分析

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部 意见反馈