| PMA-实时荧光PCR快速检测蔬果中金黄色葡萄球菌活菌 |
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| 引用本文： | 於颖,王文静,陆晔,张曦,张红芝,顾其芳,陈敏. PMA-实时荧光PCR快速检测蔬果中金黄色葡萄球菌活菌[J]. 现代预防医学, 2016, 0(20): 3757-3763 |
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| 作者姓名： | 於颖 王文静 陆晔 张曦 张红芝 顾其芳 陈敏 |
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| 作者单位： | 上海市疾病预防控制中心，上海 200336 |
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| 摘 要： | 目的 将叠氮溴化丙锭（PMA）与实时荧光定量PCR（real-time PCR）技术相结合，快速检测蔬果中金黄色葡萄球菌活菌。方法 通过对影响PMA作用的关键因素，包括PMA浓度、光照时间以及活、死菌比例混合等进行试验，确定PMA的作用条件。以金黄色葡萄球菌nuc基因为靶基因，建立PMA-实时荧光定量PCR方法，并构建金黄色葡萄球菌重组质粒标准品，用于检测蔬果中金黄色葡萄球菌活菌。结果 在PMA 30 μg/ml浓度下，曝光5 min，可实现PMA筛选金黄色葡萄球菌活菌的作用。由构建的质粒标准品模板建立标准曲线表现出良好线性关系，相关系数r = 0.9995，所建立的方法灵敏度可达到14 copies /μl，且特异性良好。经2 h增菌后用PMA进行筛选，再用荧光定量PCR检测，可实现快速对蔬果样品中活的金黄色葡萄球菌的检测。结论 用PMA-实时荧光定量PCR方法可快速检测蔬果中活的金黄色葡萄球菌，可为食品安全风险监测提供可靠数据。
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| 关 键 词： | 叠氮溴化丙啶 荧光定量PCR 金黄色葡萄球菌 活菌 蔬果 |
Rapid detection of viable Staphylococcus aureus in vegetables and fruits using PMA-real time PCR |
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| YU Ying,WANG Wen-jing,LU Ye,ZHANG Xi,ZHANG Hong-zhi,GU Qi-fang,CHEN Min. Rapid detection of viable Staphylococcus aureus in vegetables and fruits using PMA-real time PCR[J]. Modern Preventive Medicine, 2016, 0(20): 3757-3763 |
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| Authors: | YU Ying WANG Wen-jing LU Ye ZHANG Xi ZHANG Hong-zhi GU Qi-fang CHEN Min |
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| Affiliation: | Shanghai Municipal Center for Disease Control and Prevention, Shanghai 200336, China |
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| Abstract: | Objective The aim of this study was to use propidium monoazide (PMA) combined with real-time fluorescent quantitative polymerase chain reaction (RT-quantitative PCR) to rapid detect viable Staphylococcus aureus in fruits and vegetables. Methods To optimize the function of PMA, detailed experiments were conducted to look into the key factors affecting the efficiency of PMA including dose concentration, exposure time to light, and live/dead bacteria cells. S. aureus nuc gene was used as our target to establish the method of PMA-RT-PCR. Furthermore, standard plasmid was also constructed for the detection of viable S. aureus in fruits and vegetables. Results With a concentration of 30μg/mL, and 5 min exposure time, PMA can be used for screening viable S. aureus. Standard curves established by plasmid standards showed good linear relation, of which the correlation coefficient r approached 0.9995. The sensitivity of this method was up to 14 copies/μL with good specificity. After a 2 h enrichment, live cells in fruits and vegetables could quickly be detected by PMA screening followed by RT-PCR amplification. Conclusion PMA-RT-PCR method could rapidly detect viable S. aureus in fruits and vegetables, providing reliable data for the food safety risk monitoring. |
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| Keywords: | Propidium monoazide Real-time PCR S. aureus Viable bacterium Fruits and vegetables |
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