Rapid and sensitive detection of Enterobacter sakazakii by cross-priming amplification combined with immuno-blotting analysis
Rapid and sensitive detection of Enterobacter sakazakii by cross-priming amplification combined with immuno-blotting analysis
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DOI：
10.1016/j.mcp.2010.09.001
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发表时间：
2010-12-01
影响因子：
3.3
通讯作者：
Huang Xitai
中科院分区：
文献类型：
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作者：
Zhao Yulong;Zhang Xia;Huang Xitai
阪崎肠杆菌是一种分布广泛、危害生命的细菌，特别是在受污染的婴幼儿配方奶粉中，阪崎肠杆菌的检测方法有生理生化法、聚合酶链式反应和环介导等温扩增等，但这些方法存在检测时间长、灵敏度低或使用有毒试剂等缺点。我们采用等温交叉引物法和免疫印迹分析相结合的方法，使整个检测过程更灵敏、更省时。根据阪崎肠杆菌16S-23S rDNA内转录间隔区的6个特异序列，设计了一套特异的置换引物、交叉引物和检测引物。CpA的特异性扩增和杂交步骤同时进行，在54个不同的细菌和236个奶粉产品的小组中检测CPA的特异性。所有阳性的阪崎肠杆菌在BioHelix Express条带上都出现了两条红色信号线，而非E sakazakii菌株只有一条信号线。对于纯细菌培养，CPA的检测限为63+/-2 7277fg，对纯菌培养的检测限为+/-8 7892 cfu/ml，预富集后为3 2+/-2 0569 cfu/100g奶粉。CPA法结合免疫印迹法是一种特异、灵敏的快速检测E sakazakii(C)2010 Elsevier Ltd.的方法，版权所有
Enterobacter sakazakii is a widespread and life-threatening bacterium especially in polluted powdered Infant milk formula Several methods have been developed for detection of E sakazakii such as physiological and biochemical methods PCR and loop-mediated isothermal amplification However these procedures were disadvantages due to a long assay time low sensitivity or the use of toxic reagents Our method of cross-priming amplification (CPA) under isothermal conditions combined with immunoblotting analysis made the whole detection procedure more sensitivity and lower time-consuming A set of specific displacement primers cross primers and testing primers were designed based on six specific sequences in E sakazakii 16S-23S rDNA internal transcribed spacer Under isothermal condition at 63 degrees C for 60 min the specific amplification and hybridization steps were processed simultaneously The specificity of the CPA was tested in panel of 54 different bacterial strains and 236 milk powder products Two red signal lines were developed on the BioHelix Express strip in all of positive E sakazakii strains and only one signal line was demonstrated by non- E sakazakii bacterial strains The limit of decetion of CPA was 63 +/- 2 7277 fg for the genomic DNA 88 +/- 8 7892 cfu/ml for pure bacterial culture and 3 2 +/- 2 0569 cfu per 100 g milk powder with pre-enrichment The current study demonstrated that the assay method of CPA combined with immuno-blotting analysis was a specific and sensitive detection for the rapid detection of E sakazakii (C) 2010 Elsevier Ltd All rights reserved