Cloning and Sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula
Cloning and Sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula
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DOI：
10.1128/aem.72.4.2539-2546.2006
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发表时间：
2006-04-01
影响因子：
4.4
通讯作者：
Venkitanarayanan, KS
中科院分区：
文献类型：
--
作者：
Nair, MKM;Venkitanarayanan, KS
坂崎肠杆菌是一种新兴的婴儿配方奶粉传播的病原体，可引起新生儿和婴儿严重的脑膜炎、脑膜脑炎、败血症和坏死性小肠结肠炎，致死率高。传统的检测方法需要长达7天的时间来识别E。坂崎。外膜蛋白A（ompA）基因沿着序列来自大肠杆菌（E.将sakazakii（ATCC 51329）克隆到pGEM-T Easy载体中并测序。与GenBank数据库中的其他序列的ompA基因的核苷酸和推导的氨基酸序列的比较，揭示了属于肠杆菌科的其他革兰氏阴性菌的ompA基因的高度同源性。根据E. sakazakii，合成两条引物，建立并优化了一条E.坂崎特异性PCR。PCR扩增出469 bp的DNA片段。sakazakii菌株，但不是来自其他细菌。实验表明，该方法能检测出10（3）CFU/ml的大肠杆菌。直接在婴儿配方食品中检测坂崎氏菌，8小时富集步骤后检测10（-1）CFU/ml。我们的结论是，这种PCR，结合富集培养，有可能被用作一种快速的工具，用于检测是否存在E。婴儿配方奶粉中的坂崎。
Enterobacter sakazakii is an emerging, infant formula-borne pathogen that causes severe meningitis, meningoencephalitis, sepsis, and necrotizing enterocolitis in neonates and infants, with a high fatality rate. Traditional detection methods take up to 7 days to identify E. sakazakii. The outer membrane protein A gene (ompA) along with its flanking sequences from E. sakazakii (ATCC 51329), was cloned in the pGEM-T Easy vector and sequenced. Comparison of the nucleotide and deduced amino acid sequences of the ompA gene with other sequences available in the GenBank database revealed a high degree of homology with ompA genes of other gram-negative bacteria belonging to the Enterobacteriaceae. Based on regions of the ompA gene unique to E. sakazakii, two primers were synthesized to develop and optimize an E. sakazakii-specific PCR. The PCR amplified a 469-bp DNA product from all E. sakazakii strains tested but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 10(3) CFU/ml of E. sakazakii bacteria in infant formula directly and 10(-1) CFU/ml after an 8-h enrichment step. We conclude that this PCR, combined with enrichment culturing, has the potential to be used as a rapid tool for detecting the presence of E. sakazakii in infant formula.