肺炎克雷伯菌环介导等温扩增技术检测方法的建立

doi:10.16431/j.cnki.1671-7236.2018.11.004

摘要/Abstract

摘要：

本研究旨在建立一种环介导等温扩增技术（loop-mediated isothermal amplification，LAMP）检测食品中的肺炎克雷伯菌。选取肺炎克雷伯菌特有的毒力基因区域，设计2对引物（1对内引物、1对外引物），进行体外恒温扩增反应。对反应体系内的甜菜碱、核酸原料（dNTPs）、内外引物比例、Mg²⁺浓度、反应时间和温度进行优化和选择，并进行特异性和灵敏性试验检测。应用建立的LAMP方法对灭菌鸡肉人工污染加标样品进行检测。结果表明，通过优化试验，筛选到最优反应体系和反应条件；应用优化后的LAMP反应体系对包括肺炎克雷伯菌在内的18种常见微生物进行特异性检测试验，特异性良好。肺炎克雷伯菌LAMP反应体系对基因组的最低检测限是0.875 pg/μL，比传统PCR方法灵敏性高100倍。对人工鸡肉阳性样品进行检测，检测限为30 CFU/mL。本试验成功建立了一种能检测食物中致病肺炎克雷伯菌的LAMP检测方法，该方法利用2对引物针对肺炎克雷伯菌目的基因6个区域进行体外恒温核酸阶梯性扩增，灵敏性更高，反应时间更短，检测成本更低。

关键词: 肺炎克雷伯菌; 环介导等温扩增(LAMP); 检测

Abstract:

This study was aimed to establish a loop-mediated isothermal amplification (LAMP) to detect Klebsiella pneumoniae in food.The virulence gene region unique to Klebsiella pneumoniae was selected,and two pairs of primers (one pair of inner primers and one pair of external primers) were designed to perform an in vitro constant temperature amplification reaction.The betaine,nucleic acid material (dNTPs),internal and external primer ratio,Mg²⁺ concentration,reaction time and temperature in the reaction system were optimized and selected,and specificity and sensitivity tests were performed.At the same time,the established LAMP method was used to detect the artificial contamination of the sterilized chicken.Through optimization experiments,the optimal reaction system and reaction conditions were screened.Using the optimized LAMP reaction system,specific detection tests were performed on 18 common microorganisms including Klebsiella pneu- monia,the results showed that the method was specific.The minimum detection limit for the genome of Klebsiella pneumonia LAMP reaction system was 0.875 pg/μL,which was 100 times more sensitive than traditional PCR method.The artificial chicken positive samples were tested and the detection limit was 30 CFU/mL.This experiment successfully established a LAMP detection method for detecting pathogenic Klebsiella pneumonia in food.Two pairs of primers were used to thermostatically amplify the target DNA in 6 regions of Klebsiella pneumoniae with staircase product in vitro,which was more sensitive with shorter reaction time and lower testing cost.

Key words: Klebsiella pneumoniae; loop-mediated isothermal amplification (LAMP); detection

中图分类号:

徐云明, 魏利斌, 周弇扬, 李叶辉, 孙智远, 仲思远, 陈爱宁, 郭丽娟, 柳增善, 任洪林. 肺炎克雷伯菌环介导等温扩增技术检测方法的建立[J]. 《中国畜牧兽医》, 2018, 45(11): 3003-3010.

XU Yunming, WEI Libin, ZHOU Yanyang, LI Yehui, SUN Zhiyuan, ZHONG Siyuan, CHEN Aining, GUO Lijuan, LIU Zengshan, REN Honglin. Establishment of Loop-mediated Isothermal Amplification (LAMP) Method for Klebsiella pneumoniae[J]. , 2018, 45(11): 3003-3010.

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