【摘要】 目的:建立实时荧光TaqM anTM定量检测蜡样芽胞杆菌的快速检测方法,为食品中蜡样芽胞杆菌污染的调查及食物中毒的快速准确定量检测提供手段。方法:通过分析蜡样芽胞杆菌16S rDNA、23S rDNA、16S-23S ITS、gyrB、groEL、cereolysin AB、HBL(hb lA、hb lC、hb lD)、NHE(nheA、nheC、nheD)、bce-T等目的基因,对比所选取的不同目的基因优缺点,最终选择合适的目的基因16S-23S ITS进行同源性分析,并根据GenBank公布的蜡样芽胞杆菌16S-23S ITS基因的保守序列,用引物与探针专业设计软件自行设计引物和TaqM anTM探针,并利用硅胶模型TM基因组DNA提取法制备DNA标准品,通过对TaqM anTM实时荧光PCR退火温度、探针浓度、引物浓度、Mg2+离子浓度、缓冲液浓度及酶用量等反应条件的优化,初步建立蜡样芽胞杆菌的实时荧光TaqM anTM快速定量检测方法,并利用该方法对模拟样品进行检测。结果:选取的16S-23S ITS基因同源性达到99%以上,特异性好;所有蜡样芽胞杆菌均含有两段16S-23S ITS基因,有利于提高反应的灵敏度。通过对各实验条件的优化,得出蜡样芽胞杆菌TaqM anTM实时荧光PCR的最佳反应条件,初步建立起实时荧光TaqM anTM定量检测蜡样芽胞杆菌的快速检测方法,该方法能在3 h左右完成整个检测过程。结论:实时荧光Taq-M anTM定量检测法不但灵敏度好,而且特异性高,检测时间短,能提高蜡样芽胞杆菌的检出率和检测准确性,可望应用于蜡样芽胞杆菌食物中毒的快速准确定量诊断。更多还原

【Abstract】 Objective:To establish a rapid TaqMan^TM real-time PCR assay for the quantitative detection of Bacillus cereus,an alternative method for the rapid detection of Bacillus cereus caused food borne disease.Methods:Through analyzing 16S rDNA,23S rDNA,16S-23S ITS,gyrB,groEL,cereolysin AB,HBL（hblA,hblC,hblD）,NHE（nheA,nheC,nheD） and bceT,et al,16S-23S ITS was chosen as target gene and do alignment analysis.To prepare DNA standard,DNA template was extracted by using silica gel model TM genome DNA extraction and quantified.One set of primers and probe were designed based on the core sequence of ITS gene published in the GenBank.And it was applied to develop the rapid TaqMan^TM real-time assay for Bacillus cereus.Results:The alignment of 16S-23S ITS was up to 99%,and the gene 16S23S ITS was in favor of enhancing the sensitivity of PCR reaction because it′s a two copy gene.Through optimizing the reaction system and conditions,the established method could be applied to detect the number of Bacillus cereus in the samples in about 3 hours.Conclusion:TaqMan^TM real-time assay is rapid,sensitive and specific.It could be applied to the rapid diagnosis of Bacillus cereus caused food poisoning.更多还原