实验动物金黄色葡萄球菌LAMP检测方法的建立  

Establishment of a Loop-Mediated Isothermal Amplification Technique for Detecting Staphylococcus aureus in Laboratory Animals

作　　者：荣蓉[1] 张丽芳[1] 李雨函[1] 佟巍[1] 刘先菊[1] 王艳蓉[1] 向志光[1] 刘云波[1] 

机构地区：[1]中国医学科学院医学实验动物研究所北京协和医学院比较医学中心卫生部实验动物检测中心,北京100021

出　　处：《中国比较医学杂志》2012年第4期68-72,共5页Chinese Journal of Comparative Medicine

基　　金：中央级公益性科研院所基本科研业务费专项资金项目(DWS1106)

摘　　要：目的建立一种能够应用于实验动物金黄色葡萄球菌检测及鉴定的环介导等温扩增(loop-mediatedisothermal amplification,LAMP)方法。方法本研究针对金黄色葡萄球菌特异的nuc基因设计4条引物,对反应条件进行优化。结果通过优化,该方法 60℃、40 min、Mg2+浓度8 mmol/L、dNTP浓度2.0 mmol/L、甜菜碱浓度0.8mmol/L时,对金黄色葡萄球菌检测限为2 cfu,且与其它常见实验动物致病菌无交叉反应。反应结果可通过添加荧光染料SYBR green I直接肉眼观察。结论本研究建立的金黄色葡萄球菌LAMP方法具有快速、灵敏、操作简单、设备要求低的特点,适用于基层、大批量样本、快速检测的要求。Objective To establish a loop-mediated isothermal amplification（LAMP） technique for detecting Staphylococcus aureus in laboratory animals.Methods Four primers were designed and screened specifically to recognize the nuc gene of Staphylococcus aureus.Optimal reaction conditions were screened.Results 2 cfu extracted crude DNA and no cross reaction with other commonly seen pathogenic bacteria was found when the conditions were 60℃ for 40 min at least,Mg2＋at a concentration of 8.0 mmol/L,dNTP 2.0 mmol/L and betaine 0.8mmol/L.The result of reaction can be directly observed with naked eye by adding a fluorescent SYBR green I dye.Conclusions This LAMP technique for detectiong Staphylococcus aureus is sensitive,rapid and simple to perform,and can satisfy the requirements of grassroots lab,mass samples and quick detection.

关 键 词：LAMP 金黄色葡萄球菌 检测 

分 类 号：R33[医药卫生—人体生理学]