荧光定量PCR检测金黄色葡萄球菌肠毒素A方法研究  

Development of fluorescence quantitative PCR method for detection of enterotoxin A of Staphylococcus aureus

作　　者：刘渠[1] 廖灵灵[2] 徐亚军[1] 李涌[2] 

机构地区：[1]广东省深圳市龙岗区疾病预防控制中心,广东深圳518172 [2]四川大学华西公共卫生学院,成都610041

出　　处：《中国卫生检验杂志》2010年第4期788-789,803,共3页Chinese Journal of Health Laboratory Technology

摘　　要：目的:建立快速、灵敏、特异地检测金黄色葡萄球菌肠毒素A的荧光定量PCR检测方法。方法:以SEA基因作为肠毒素A的检测靶序列,设计荧光定量PCR引物和Taqman探针,优化反应体系的主要成分浓度和循环条件,评价方法的灵敏度、特异性和重复性,并对22株食物中毒中分离的金黄色葡萄球菌进行肠毒素A的检测。结果:建立金黄色葡萄球菌肠毒素A的荧光定量PCR检测方法。25μl荧光定量PCR反应体系主要成分浓度分别为Mg2+7.0 mmol/L,dNTP 0.3 mmol/L,引物0.4μmol/L,探针0.7μmol/L,TaqDNA聚合酶2.5U。方法可检出最低45个拷贝的细菌DNA,特异性和重复性良好。从22株食物中毒中分离的金黄色葡萄球菌中检出肠毒素A阳性菌株6株。结论:荧光定量PCR可快速、准确的检出金黄色葡萄球菌的肠毒素A,能为金黄色葡萄球菌食物中毒诊断提供依据。Objective：To establish a fluorescence quantitative PCR assay which was specific and sensitive for rapidly detecting Staphylococcal enterotoxin A.Methods：The SEA gene was selected to be the target gene to design and synthesize the tagman probe for the primer.The PCR system conditions were optimized.Then the sensitivity,specificity and repeatability of the method were evaluated.22 isolates Staphylococcus aureus from food posioning sample were analyzed.Results：The fluorescence quantitative PCR method was well established.The best 25 μl reaction components was 7.0 mmol/L Mg2＋,0.3 mmol/L dNTP,0.4 μmol/L primers,0.7 μmol/L probe and 2.5U TaqDNase.The detection limit for staphylococcus aureus enterotoxin A was 45 copies.Among 22 samples,6 strains were positive for enterotoxins A.Conclusion：The TaqMan PCR could rapidly and exactly detect the staphylococcus aureus enterotoxins A,which can provide evidence for the rapid diagnosis of poisoning patients and symptomatic treatment.

关 键 词：金黄色葡萄球菌 肠毒素A 荧光定量PCR 

分 类 号：R446.5[医药卫生—诊断学]