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[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 a method for the rapid detection of Enterobacter sakazakii by immunomagnetic beads adsorption-fluorescence quantitative PCR (IMB-FPCR) and to explore the application in the rapid diagnosis of dairy product contamination monitoring and food poisoning. [Method] According to the sequence of rpsU-dnaG gene of Enterobacter sakazakii, primers and probes were designed and used to construct the quantitative standard for the detection of Staphylococcus aureus using gene recombination technology. The detection method of Enterobacter sakazakii IMB-FPCR was established. [Result] The Enterobacter sakazakii recombinant plasmid standard and the E. sakazakii IMB-FPCR method were successfully constructed. Enterobacter sakazakii immune magnetic beads adsorption test showed that the magnetic beads in milk powder Enterobacter sakazakii adsorption rate of 77.7%. The results of specificity, sensitivity, stability and repeatability showed that the bacterial cells with better specificity and the lowest detection limit of 10 cfu / ml had good stability and repeatability. The entire test only takes 1d. [Conclusion] The IMB-FPCR is simple, rapid, sensitive and specific. It provides a new way to accurately detect Enterobacter sakazakii in dairy products. It can be widely used in food hygiene supervision, commodity inspection and quarantine, and clinical diagnosis.