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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 30μg/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。
Objective To rapidly detect Staphylococcus aureus viable cells by combining azidium bromide (PMA) with real-time PCR (real-time PCR). Methods The key factors influencing PMA, including the concentration of PMA, light time and the mixture ratio of live and dead bacteria, were tested to determine the working conditions of PMA. Staphylococcus aureus nuc gene as target gene, the establishment of PMA-real-time fluorescence quantitative PCR method, and the construction of Staphylococcus aureus recombinant plasmid standard for the detection of Staphylococcus aureus live in fruits and vegetables. Results The effect of PMA screening of viable Staphylococcus aureus was achieved by exposure to PMA 30 μg / ml for 5 min. The established standard curve of the plasmid standard template showed a good linear relationship, the correlation coefficient r = 0.9995, the sensitivity of the established method can reach 14 copies / μl, and the specificity is good. After 2 h of enrichment with PMA screening, and then detected by real-time PCR, which can achieve rapid detection of Staphylococcus aureus live in fruits and vegetables samples. Conclusion The rapid detection of live S. aureus in fruits and vegetables by PMA-real-time fluorescence quantitative PCR method can provide reliable data for food safety risk monitoring.