荧光定量PCR检测金黄色葡萄球菌方法的建立及应用  

Establishment and application of the real-time fluorescence-based quantitative PCR method for detection of Staphylococcus aureus

作　　者：郭梦冉[1] 董兵 李聪[1] 李志辉 王庆平[1] 张伟 檀建新[1] GUO Meng-ran;DONG Bing;LI Cong;LI Zhi-hui;WANG Qing-ping;ZHANG Wei;TAN Jian-xin(College of Food Science and Technology,Engineering Research Center of Hebei Province for Agricultural Products Processing,Hebei Agricultural University,Baoding 071001,China;College of Science and Technology,Hebei Agricultural University,Huanghua 061100,China)

机构地区：[1]河北农业大学食品科技学院/河北省农产品加工工程技术中心,河北保定071001 [2]河北农业大学理工学院,河北黄骅061100

出　　处：《河北农业大学学报》2018年第3期72-76,83,共6页Journal of Hebei Agricultural University

基　　金：河北省重点研发计划项目(16275505D);河北省食品科学与工程学科"双一流"建设资金项目(2016SPGCA18);国家自然基金项目(31371772)

摘　　要：为了检测食品中金黄色葡萄球菌,建立一种基于SYBR GreenⅡ染料的实时荧光定量聚合酶链式反应(qPCR)检测方法。根据金黄色葡萄球菌nuc基因,设计特异引物,优化引物退火温度,检测了特异性和灵敏性,建立了荧光定量PCR检测方法,并利用此方法对肉类和乳制品中的金黄色葡萄球菌进行检测。结果表明:建立qPCR方法具有很好的特异性,其灵敏度可达到3.5×101 CFU/mL,检测到的循环阈值(Cycle threshold,Ct)与金黄色葡萄球菌菌液浓度有良好的线性方程,相关系数R2为0.9997,在肉类和乳制品中金黄色葡萄菌的检测限分别为4.0×101,3.5×101 CFU/mL。试验证明建立的荧光定量PCR检测方法能够快速准确地检测食品中的金黄色葡萄球菌。In order to detect Staphylococcus aureus in food, a real-time fluorescence-based quantitative PCR （qPCR） detection method based on SYBR Green II dye was established. According to the S. aureus nuc gene, the specific primers were designed, The annealing temperature was optimized and the specificity and sensitivity of the qPCR method for detection of S. aureus were investigated. The detection limit of this method was tested in meat and dairy products. Results indicated that with perfect specificity, the detection sensitivity of the qPCR method was 3.5×10^ CFU/mL. There was a perfect linear equation between the Ct value and the concentration of S. aureus . The correlation coefficient R^ was 0.9997. The detection limit of S. aureus in meat and dairy products was 4.0×10^1 CFU/mL and 3.5×10^1 CFU/mL, respectively. Experiments showed that the qPCR detection method established in this study can be applied to quickly and accurately detect S. aureus in food.

关 键 词：荧光定量PCR 金黄色葡萄球菌 肉类 乳制品 

分 类 号：R155.5[医药卫生—营养与食品卫生学]