荧光定量PCR快速检测金黄色葡萄球菌方法的建立  

Establishment of real-time quantitative PCR-based methods for detection of Staphylococcus aureus in food

作　　者：苏裕心[1,2] 高姗[2] 康琳[2] 赵耀[2] 郑学礼[1] 王景林[2] 

机构地区：[1]南方医科大学公共卫生与热带医学学院病原生物学系,广州510515 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学科学院院刊》2010年第1期25-29,39,共6页Bulletin of the Academy of Military Medical Sciences

基　　金：国家科技支撑计划(2008BAK41B03)

摘　　要：目的利用SmartCycler系统建立一种简便、快速的荧光定量PCR检测金黄色葡萄球菌(Staphylococcus aureus,简称SAU)的方法。方法根据SAU特异nuc基因设计引物和探针,建立检测体系,并在体系中加入内质控IAC,通过另一个标记不同荧光基团的TaqMan探针来监测IAC。用SAU6538株污染饮用水和牛奶模拟实际样本,以评价体系的性能。结果以含nuc片段的线性质粒为模板,反应体系的灵敏度为5拷贝/μl;以6538株基因组DNA为模板,最低检测限为10fg/μl;用涂平板法计数并10倍梯度稀释的菌液提取DNA为模板,最低检测限为50cfu/ml。建立的定量标准曲线的循环域值(Ct)与模板拷贝数呈良好的线性关系(r2≥0.998),PCR效率E≥96.7%。用SAU6538污染饮用水和牛奶模拟实际样本,灵敏度可达5×102cfu/25ml样本。结论所建立的nuc-IAC荧光定量PCR具有内质控IAC,既能定量检测食物是否被污染,又能监测PCR体系,防止假阴性发生或低估病原菌污染量。Objective To develop a sensitive,specific,simple and rapid quantitative real-time PCR （Q-PCR） assay for detection of Staphylococcus aureus with SmartCycler.Methods According to the nuc gene sequences specific to S.aureus,a pair of primers and one TaqMan probe were designed.An internal amplification control （IAC） which is a chimeric double-stranded DNA constructed from a fragment of the Listeria monocytogenes hly gene flanked by the nuc-specific target sequences was added to the reaction system.This IAC was detected using a second TaqMan probe labeled with a different fluorophore.The performance of the nuc-IAC Q-PCR was evaluated using artificially contaminated drinking water and commercial UTH whole milk samples spiked with ATCC 6538.Results The nuc-IAC assay could be used reliably for detection with a sensitivity of 5 copies of linear plasmid DNA per reaction,10 fg of genomic DNA in 62.5% of the reactions or 50 cfu/ml S.aureus cells with 50% probability.The quantification was linear （r2≥0.998） over a 6-log dynamic range,with a PCR efficiency over 0.967.The 5×102 CFU per 25 ml mimic sample of drinking water or milk could be detected by this assay consistently and quantifiably.Conclusion The nuc-IAC Q-PCR assay for S.aureus is developed.It could not only be applied for the quantitative detection of S.aureus,but also prevent the false negatives and underestimations of contamination loads due to PCR failure.

关 键 词：金黄色葡萄球菌 实时定量PCR 病原体检测 饮用水 牛奶 

分 类 号：R450[医药卫生—治疗学]