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

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.