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当前位置: 首页 > 文章 > 荧光定量PCR检测生鲜猪肉中金黄色葡萄球菌的应用研究 河南农业大学学报 2014,48 (4) 475-480
Position: Home > Articles > Application of real-time fluorescent PCR detection of Staphylococcus aureus in fresh pork Journal of Henan Agricultural University 2014,48 (4) 475-480

荧光定量PCR检测生鲜猪肉中金黄色葡萄球菌的应用研究

作  者：
李苗云;惠利娜;赵改名;刘杰;黄现青;高晓平
单  位：
河南省肉制品加工与质量安全控制重点实验室;河南农业大学食品科学技术学院
关键词：
荧光定量PCR;金黄色葡萄球菌;生鲜猪肉;应用研究
摘  要：
以金黄色葡萄球菌的耐热核酸酶基因nuc为靶基因,设计并筛选引物,建立并优化金黄色葡萄球菌的荧光定量PCR检测方法,结合BP平板计数法,建立20℃条件下生鲜猪肉中金黄色葡萄球菌的生长曲线.优化建立的反应体系为:2×SYBR Premix Ex TaqTM 10μL,上、下游引物(1μmol)各2.0μL,模板为2.0μL,水4μL共20μL;反应条件为:95℃预变性30 s;95℃变性5 s,63℃退火34 s,共40个循环.与BP平板计数法的检测结果拟合后均呈现典型"S"型生长曲线.在生长曲线的延滞期和对数期,2种方法检测结果符合率为100%.进入稳定期后,荧光定量PCR检测结果高于传统平板计数.荧光定量PCR比传统细菌检测方法更特异、快速、灵敏.
译  名：
Application of real-time fluorescent PCR detection of Staphylococcus aureus in fresh pork
作  者：
LI Miao-yun;HUI Li-na;ZHAO Gai-ming;LIU Jie;HUANG Xian-qing;GAO Xiao-ping;College of Food Science and Technology,Henan Agricultural University,Henan Key Laboratory of Meat Processing and Quality Safety Control;
关键词：
real-time fluorescent PCR;;Staphylococcus aureus;;fresh pork;;application
摘  要：
A real-time fluorogenic PCR was developed for the reliable identification of Staphylococcus aureus strains harboring the thermostable nuclease gene( nuc) by designing and screening primer. The growth curves of Staphylococcus aureus in the fresh pork was established under the condition of 20 ℃through traditional plate count method and the real-time fluorogenic PCR assays. The results showed that optimizational reaction system and reaction conditions of the real-time fluorogenic PCR were 2 ×SYBR Premix Ex TaqTM 10 μL,each of upstream primer and downstream primer( 1 μmol) 2 μL,DNA sample 2 μL,sterile water 4 μL,a total of 20 μL; pre-denaturing for 30 s at 95 ℃,denaturing for 5 s at 95 ℃,annealing primers for 34 s at 63 ℃,using 40 cycles. Both of them took on the typical"S"type growth curve. The results of the two approaches were accordant in the lag-phase and logphase of the growth curve,but in the stable-phase the result of the real-time fluorescence PCR assays was significantly higher than that of the plate count method. The real-time fluorogenic PCR resulted in a more specificity,celerity,sensitivity. Therefore,during the shelf life of fresh pork,the real-time fluorogenic PCR was suitable for the fast quantitative detection of Staphylococcus aureus strains.

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