实时荧光环介导等温扩增技术检测乳粉中的肺炎克雷伯氏菌

为了建立食品中肺炎克雷伯氏菌灵敏快速的检测方法,根据GenBank中已公布的肺炎克雷伯氏菌phoE基因设计引物,利用实时荧光监测仪,建立实时荧光环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术检测食品中肺炎克雷伯氏菌的方法,可直观判定检测结果,仪器亦可自动显示检测结果。对该检测方法的特异性、灵敏度等方面进行研究,并与聚合酶链反应(polymerase chain reaction,PCR)检测方法进行比较。结果表明,实时荧光LAMP检测方法特异性强、灵敏度高。用于特异性实验的21株实验菌株中,4株肺炎克雷伯氏菌,呈现阳性结果,而其他17株非肺炎克雷伯氏菌,均呈阴性结果;检测纯菌的灵敏度和检测人工污染乳粉的检出限分别为79 CFU/mL和79 CFU/g,是常规PCR检测方法的100倍。总之,本研究建立的实时荧光LAMP检测方法灵敏度高、特异性强、耗时短、结果判定直观,实现了对肺炎克雷伯氏菌的快速检测。

Detection of Klebsiella pneumoniae in Milk Powder by Real-Time Fluorescence Loop-Mediated Isothermal Amplification Method

Klebsiella pneumoniae which can cause diseases such as pneumonia was identified as a new foodborne
pathogenic bacterium in recent years. Using an ESE-Quant tube scanner, a new real-time fluorescent loop-mediated
isothermal amplification method for detecting K. pneumoniae was established with the primers designed based on the phoE
gene sequence published in GenBank. The results showed that this method had high specificity and sensitivity than PCR
method. Among 21 tested strains, 4 were identified as K. pneumoniae, and the other 17 were negative. The detection limit of
K. pneumoniae in artificially contaminated milk powder was 79 CFU/g, 100 times more sensitive than the conventional PCR
method. Moreover, the positive results could be directly identified immediately through the data and chart collected by the
ESE-Quant tube scanner so that real-time results could be provided.