人类特异性粪厌氧菌基因标记物实时荧光定量PCR检测方法的建立和应用  

Development and application of real-time fluorescence quantitative PCR detection method of human specific genetic markers from fecal anaerobes

作　　者：李宪[1] 张崇淼[1] 王晓昌[1] 孙婷婷[1] 谢润欣[1] 

机构地区：[1]西安建筑科技大学环境与市政工程学院,西北水资源与环境生态教育部重点实验室,西安710055

出　　处：《环境工程学报》2013年第1期384-388,共5页Chinese Journal of Environmental Engineering

基　　金：国家自然科学基金资助项目(50908185);中国博士后基金项目(20100470086);陕西省教育厅专项科研项目(11JK0765)

摘　　要：针对人类粪便污染,选择来源于拟杆菌和双歧杆菌16S rRNA的基因片段作为标记物,分别建立了相应的实时荧光定量PCR检测方法。选取不同来源的粪便样品进行检测,证实了引物的特异性。回收纯化从污水中扩增所得的目标PCR片段,经过连接转化,筛选阳性克隆提取其重组质粒作为实时荧光定量PCR的标准品。通过检测一系列梯度稀释的标准品,确定了拟杆菌基因标记物和双歧杆菌基因标记物定量PCR检测方法分别在1.98×102～1.98×107copy/μL和2.12×101～2.12×107copy/μL范围内具有良好的线性关系。For the human feces pollution, based on 16S rRNA gene fragments from bacteroidales and bifidobacterium, corresponding real-time fluorescence quantitative polymerase chain reaction （real-time PCR） test methods were established. Choosing different sources of feces samples for testing, the primer specificity was confirmed. After recovering the purification of target PCR segments from sewage, through connection and trans- formation, the positive cloning restructuring plasmid as real-time PCR standard product was obtained. Using a series of 10-fold dilution of recombinant plasmid DNA, the bacteroidales and bifidobacterium genetic markers presented good linear relationship respectively in 1.98 × 102 - 1.98 × 107 copy/μL and 2. 12 × 101 - 2.12 × 107 copy/μL.

关 键 词：人类特异性 粪厌氧菌 基因标记物 实时荧光定量PCR 

分 类 号：X502[环境科学与工程—环境工程]