免疫磁珠与荧光定量PCR联合检测乳制品中阪崎肠杆菌的实验研究  

RAPID DETECTION OF ENTERBACTER SAKAZAKII IN DAIRY USING IMMUNOMAGNETIC BEAD COMBINED WITH REAL TIME FLUORESCENCE QUANTITATIVE PCR

作　　者：杨柳[1] 苏明权[1] 马越云[1] 焦刚 常亮[1] 肖凤静[1] 李明 彭年才 郝晓柯[1] 

机构地区：[1]第四军医大学西京医院全军临床检验医学中心,西安710032 [2]西安天隆科技有限公司

出　　处：《现代预防医学》2011年第6期1086-1089,共4页Modern Preventive Medicine

摘　　要：[目的]建立一种免疫磁珠吸附-荧光定量PCR(IMB-FPCR)快速检测阪崎肠杆菌的方法,探讨在乳制品污染监测及食物中毒快速诊断中的应用。[方法]根据阪崎肠杆菌rpsU-dnaG基因序列设计引物和探针,采用基因重组技术构建用于金黄色色葡萄球菌检测的定量标准品,建立阪崎肠杆菌IMB-FPCR检测方法。[结果]成功构建了阪崎肠杆菌重组质粒标准品和阪崎肠杆菌IMB-FPCR方法。阪崎肠杆菌的免疫磁珠吸附试验显示磁珠对奶粉中的阪崎肠杆菌吸附率达77.7%。通过特异性、敏感性、稳定性和重复性验证,结果表明具有较好的特异性,最低检测限为10cfu/ml的菌细胞,并具有良好的稳定性和重复性。整个检测仅需时1d。[结论]IMB-FPCR简便快速、灵敏度高及特异性强,为准确检测乳制品中阪崎肠杆菌提供了一个新的途径,可广泛应用于食品卫生监管、商品检验检疫以及临床诊断等。[Objective] To establish immunomagnetic bead combine with real-time fluorescent quantitative PCR （IMB- FPCR） method for rapid detecting Enterobacter sakazakii and investigate its feasibility and application value in dairy pollution monitoring. [Methods] Primers and probes were designed based on rpsU-dnaG gene sequence of Enterobacter sakazakii. Quantitative standard for Staphylococcus aureus was upbuilt by cloning of femB gene. Then IMB-FPCR for Enterobacter sakazakii was established routinely. [ Results] It was identified that rpsU-dnaG gene fragment was cloned as standard successfully. IMB adsorption of Enterobaeter sakazakii displayed that absorption rate of Enterobacter sakazakii in milk powder was 77.7%. IMB-FPCR method showed good spec/_qcity, sensit/vity, stab/lity by the verification experiment. The detect/on limit was 10cfu/ml of bacterim ceils. This method only needed one day. [Conclusion] The IMB-FPCR method is simple, rapid, and has good sensitivity and specificity, and provides a new way for accurate detection of Enterobacter sakazakii in dairy products. It could be applied to food hygiene regulation, quarantine and clinical diagnosis.

关 键 词：阪崎肠杆菌 荧光定量PCR 免疫磁珠 

分 类 号：R378.2[医药卫生—病原生物学]