金黄色葡萄球菌VBNC 状态的诱导条件和EMA-qPCR 检测

doi:10.3969/j.issn.1000-565X.2013.01.021

华南理工大学学报（自然科学版） ›› 2013, Vol. 41 ›› Issue (1): 127-132.doi: 10.3969/j.issn.1000-565X.2013.01.021

金黄色葡萄球菌VBNC 状态的诱导条件和EMA-qPCR 检测

余以刚田聪肖性龙^† 陶文扬黎金霞吴晖

华南理工大学轻工与食品学院, 广东广州 510640

收稿日期:2012-05-25 修回日期:2012-08-10 出版日期:2013-01-25 发布日期:2012-12-03
通信作者: 肖性龙(1977-),男,博士,讲师,主要从事食品安全与检测研究. E-mail:fexxl@scut.edu.cn
作者简介:余以刚(1968-),男,博士,副教授,主要从事食品安全与检测研究.E-mail:yuyigang@scut.edu.cn
基金资助:
国家自然科学基金青年科学基金资助项目(31101279);教育部高等学校博士学科点专项科研基金新教师类资助课题(20110172120034);华南理工大学中央高校基本科研业务费专项资金资助项目(2011ZM0101);广东省天然产物绿色加工与产品安全重点实验室开放课题(201101)

Induction Conditions and EMA-qPCR Detection of Staphyloccocus aureusin Viable but Non-Culturable State

Yu Yi-gang Tian Cong Xiao Xing-long Tao Wen-yang Li jin-xia Wu Hui

School of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, Guangdong, China

Received:2012-05-25 Revised:2012-08-10 Online:2013-01-25 Published:2012-12-03
Contact: 肖性龙(1977-),男,博士,讲师,主要从事食品安全与检测研究. E-mail:fexxl@scut.edu.cn
About author:余以刚(1968-),男,博士,副教授,主要从事食品安全与检测研究.E-mail:yuyigang@scut.edu.cn
Supported by:
国家自然科学基金青年科学基金资助项目(31101279);教育部高等学校博士学科点专项科研基金新教师类资助课题(20110172120034);华南理工大学中央高校基本科研业务费专项资金资助项目(2011ZM0101);广东省天然产物绿色加工与产品安全重点实验室开放课题(201101)

摘要/Abstract

摘要： 为建立金黄色葡萄球菌(Staphyloccocus aureus) 活的非可培养( VBNC) 状态的诱导条件模型,考察了温度、盐度和酸度3 个因素对金黄色葡萄球菌细菌可培养数的影响,通过正交试验优化得到了VBNC 状态的诱导条件,同时观察细菌可培养数的变化,建立了DNA 结合染料叠氮溴乙锭(EMA) 与qPCR 技术相结合检测VBNC 金黄色葡萄球菌的方法. 实验结果表明:细菌可培养数受酸度的影响最大,VBNC 状态的最佳诱导条件为菌液在含15%NaCl 和0. 3%乙酸的营养肉汤培养基中于4 ℃下培养12 d;通过正交试验诱导后的不可培养菌可由EMA-qPCR 方法有效检出,其与qPCR 法的Ct 值( 荧光信号达到设定的阈值所经历的循环数)之差在1. 29 ~8. 56 之间变化.

关键词: 金黄色葡萄球菌, 活的非可培养状态, 诱导条件, 叠氮溴乙锭, qPCR

Abstract:

In order to establish a model for the determination of induction conditions of Staphyloccocus aureus in via-ble but non-culturable (VBNC) state and to reveal the effects of temperature, salt concentration and pH value onthe culturable cell number of Staphyloccocus aureus, orthogonal experiments were carried out to optimize the inductionconditions. Then, the culturable cells were monitored and a method based on the DNA-binding dye EMA (EthidiumBromide Monoazide) and the qPCR technology was proposed to detect Staphyloccocus aureus in VBNC state. Theresults show that pH value greatly influences the culturable cell number, and that the cultivation in a nutrient brothmedium with 15% (mass fraction) NaCl and 0.3% (volume fraction) acetic acid at 4℃for 12d is best for theinduction of Staphyloccocus aureus in VBNC state. Moreover, it is indicated that EMA-qPCR is an effective methodto detect Staphyloccocus aureus in VBNC state under different induction conditions, with a Ct value improvement(cycle threshold) varying from 1.29 to 8.56.

Key words: Staphyloccocus aureus, viable but non-culturable state, induction condition, ethidium bromide monoa-zide, qPCR

余以刚田聪肖性龙陶文扬黎金霞吴晖. 金黄色葡萄球菌VBNC 状态的诱导条件和EMA-qPCR 检测[J]. 华南理工大学学报（自然科学版）, 2013, 41(1): 127-132.

Yu Yi-gang Tian Cong Xiao Xing-long Tao Wen-yang Li jin-xia Wu Hui. Induction Conditions and EMA-qPCR Detection of Staphyloccocus aureusin Viable but Non-Culturable State[J]. Journal of South China University of Technology (Natural Science Edition), 2013, 41(1): 127-132.

金黄色葡萄球菌VBNC 状态的诱导条件和EMA-qPCR 检测

Induction Conditions and EMA-qPCR Detection of Staphyloccocus aureusin Viable but Non-Culturable State

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