粪便中肠球菌SYBR GreenI荧光定量PCR检测方法的建立

Construction and primary application of SYBR GreenI real-time quantification PCR for Enterococcus

目的利用SYBR GreenI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设计合成特异性的引物;利用构建的质粒标准品绘制两种标准曲线,构建基因拷贝数、细菌数为分析指标的定量分析模型并初步应用于粪便标本的检测分析。结果所建立的SYBR GreenI荧光定量PCR方法检测灵敏度可达7个拷贝数/reaction。粪便样本根据实时荧光定量PCR方法所得的理论数值与培养菌值之间差异无显著性(P>0.05)。非炎性腹泻标本中菌数与健康成人标本中菌数差异无显著性(P>0.05)。灵敏度曲线所得的数值大于菌数标准曲线,可能由于DNA提取过程中存在部分的损失。检测粪便标本结果显示SYBR GreenI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。

Objective To consruct a molecular diagnostic method for Enterococcus based on SYBR Green I real- time quantification PCR. Methods Enterococcus genomic sequences were analyzed and speciyific primers were designed and synthesized according to the conserved gene sequence of enterococci 23S RDNA 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 SYBR GreenI real-time quantification PCR method was primarily used to feces samples test for Enterococcus Results The low detecting limit was 7 copies/reaction. Two different standand curves were dawn, thus two quantitative analysis models were built for genomic copy and CFU. Conclusions Overall,one sensitive, special and quick-performing method was established for Enterococcus;Real time quantitative PCR to detect enterococci rapidly and quantitatively in the various feces samples represents a considerable advancement and a great potential for environmental applications.