【摘要】 目的利用TaqMan荧光定量PCR方法,建立肠球菌实时荧光PCR检测方法,并初步应用于粪便中肠球菌的检测。方法根据GenBank发表的肠球菌23S rRNA基因序列的保守区域参考国外文献设计合成特异性的引物和探针[1];利用构建的质粒标准品优化Mg2+的浓度和引物探针浓度,并考核检测体系的保守性、灵敏性和重复性;初步应用于粪便标本的检测分析。结果Mg2+终浓度为4.5 mmol/L,上下游引物终浓度为0.4μmol/L,灵敏度为6拷贝数/反应;绘制两种标准曲线,构建了基因拷贝数、细菌数为分析指标的定量分析模型,检测粪便标本结果显示TaqMan荧光定量PCR方法较平板计数法敏感、快捷、简便。结论本研究建立了一种灵敏、特异、简便易行的肠球菌定量检测方法。更多还原

【Abstract】 Objective To construct a molecular diagnostic method for Enterococcus based on TaqMan real-time quantification PCR.Methods Enterococcus genomic sequences were analyzed and speciyific primers and probe were designed and synthesized according to the conserved gene sequence of enterococci 23S rRNA available in GenBank,Mg~2+ concentration and primer concentration were optimized.Sensitivity and stability analysis tests were performed by enterococcus plasmid standard substance.Two quantification analysis models were constructed.The constructed TaqMan real-time quantification PCR method was primarily used to feces samples test for Enterococcus.Results Mg~2+ concentration was optimized at 4.5 mmol/L,primer concentration was 0.4 μmol/L.Test showed that the primers and probe were highly conservative and specific.The low detecting limit was 6 copies/reaction.Two different standand curves were drawn,thus two quantitative analysis models were built for genomic copy and CFU.Primary application showed that TaqMan real-time quantification PCR method was more sensitive,easier and faster to perform the bacteria isolation and identification.Conclusions Overall,one sensitive,special and quick-performing method was established for Enterococcus;the real-time quantitative PCR to detect enterococci in the various feces samples ia a rapid and quantitative way,which represents a considerable advancement and a great potential for environmental applications.更多还原