沙门氏致病菌标准阳性模板的构建及实时荧光定量聚合酶链式反应检测  

Construction of Standard Positive Template and Development of a Real-Time Fluorescence Quantitative Polymerase Chain Reaction(FQ-PCR) Assay for Pathogenic Salmonella spp.

作　　者：陈晨[1,2] 邵彪 陈刚 黄伟东 

机构地区：[1]南通市产品质量监督检验所,江苏南通226011 [2]中国药科大学生命科学与技术学院,江苏南京211198

出　　处：《肉类研究》2014年第11期30-33,共4页Meat Research

基　　金：2013年度国家星火计划项目(2013GA690155);南通市科技计划项目(HS2012007)

摘　　要：目的:构建重组质粒作为标准阳性模板,建立食品中沙门氏致病菌实时荧光定量聚合酶链式反应检测方法。方法:以致病性沙门氏菌inv A基因上特异性片段为目标,设计并合成引物和Taq Man探针,将目标片段连接到PGM-T载体上构建重组质粒,建立实时荧光定量检测体系,并考察方法的灵敏性、特异性、重复性和准确性。结果:构建出致病性沙门氏菌特异性基因片段的重组质粒,能够作为实时荧光定量聚合酶链式反应检测方法的标准阳性模板,标准曲线方程为Y=-3.151 lg X+42.86(R2=0.999),灵敏度80拷贝反应体系,能特异区分沙门氏菌与类型的细菌,同时,批内和批间的变异系数均小于5%,具有良好的重复性。结论:本方法能够实现对食品中致病性沙门氏菌进行定性定量检测。Objective： To construct recombinant plasmids for use as standard positive template and establish a real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） assay for determining pathogenicSalmonellaspp. in foods. Methods： Primers and Taqman probe were designed and synthesized with the specific fragment of InvA gene as the target sequence. Recombinant plasmids were constructed by inserting the target gene into PGM-T carriers. Real-time fluorescent quantitative PCR method was established and its sensitivity, specificity, repeatability and accuracy were investigated. Results： The recombinant plasmids constructed using the specific sequence of pathogenicSalmonella spp. could be used as a standard positive template for fluorescent quantitative PCR. The standard curve wasY=-3.151 lgX＋42.86 （R2=0.999）, and the sensitivity of the method was 80 copies per reaction. It was specific to detect Salmonellaspp. with good repeatability （the inter-batch and intra-batch coefficients of variation were both less than 5%）. Conclusion： The FQ-PCR method allows qualitative and quantitative detection of pathogenicSalmonellaspp.

关 键 词：沙门氏致病菌 标准阳性模板 实时荧光定量聚合酶链式反应 TAQMAN探针 

分 类 号：TS207.4[轻工技术与工程—食品科学]