Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula
Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula
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DOI：
10.1016/j.mimet.2005.06.007
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发表时间：
2006-04-01
影响因子：
2.2
通讯作者：
Huang, XT
中科院分区：
文献类型：
--
作者：
Liu, Y;Cai, XN;Huang, XT
坂崎肠杆菌是引起儿童和成人脑膜炎、菌血症、败血症和坏死性小肠结肠炎的新兴病原体。以奶粉为基础的婴儿配方食品与大肠杆菌有关。在早产儿或其他免疫功能低下的婴儿中发生与坂崎相关的疫情。在这项研究中，我们建立了两个真实的时间PCR检测使用TaqMan和SYBR绿色的选择性富集后，在mLST和BHI鉴定病原体。用35株大肠杆菌对两种方法的准确性进行了检验。88株非坂崎肠杆菌。结果表明，所有E.坂崎株均为阳性，非E.坂崎菌株均为阴性。新开发的检测方法使我们能够检测1.1 CFU/100 g婴儿配方奶粉。两项检测均可以在2个工作日内完成。与传统的检测方法相比，真实的PCR方法快速、简便。本研究还开发了一种新的引物设计方法，可以用一对引物进行多次真实的PCR检测SYBR绿色。基于PCR双Tm值的检测方法更灵敏、更有效。(c)2005年Elsevier B. V.。All rights reserved.
Enterobacter sakazakii is all emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in nconates and children. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, we developed two real time PCR assays using TaqMan and SYBR Green to identify the pathogen after selective enrichment in mLST and BHI. The accuracy of two detections was tested by 35 strains of E. sakazakii and 88 non-E sakazakii bacterial strains. The results showed that all of these E. sakazakii strains were positive reaction to the detections and all of the non-E. sakazakii strains were negative. The newly developed assays enable us to detect 1.1 CFU/100 g infant formula. And both of the assays can be accomplished within 2 business days. Compared to the traditional detection, the real time PCR procedures are quicker and simpler. In this study, we also developed a new method to design the primers, which can support multiple real time PCR with one pair of primers in SYBR Green detection. The detection methods are more sensitive and effective based on Two-Tm-Value of PCR. (c) 2005 Elsevier B.V.. All rights reserved.