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

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．

Development of a loop-mediated isothermal amplification for detection of Yersinia enterocolitica in patients with diarrhea

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.