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

Abstract

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

CLC Number:

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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Establishment of Loop-mediated Isothermal Amplification (LAMP) Method for Klebsiella pneumoniae

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