| 城市污水中病原性大肠埃希氏菌毒力基因eaeA和rfbE的实时荧光定量PCR检测 |
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| 引用本文： | 谢润欣,张崇淼,王晓昌,孙婷婷,李宪.城市污水中病原性大肠埃希氏菌毒力基因eaeA和rfbE的实时荧光定量PCR检测[J].环境科学研究,2012,25(8):922-926. |
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| 作者姓名： | 谢润欣 张崇淼 王晓昌 孙婷婷 李宪 |
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| 作者单位： | 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, 陕西 西安710055 |
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| 基金项目： | 国家自然科学基金项目(50908185)；国家环境保护环境微生物利用与安全控制重点实验室开放基金项目(MARC 2011D038)；陕西省教育厅专项科研项目(11JK0765) |
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| 摘 要： | 针对病原性大肠埃希氏菌EHEC和EPEC的毒力基因eaeA和rfbE,选用特异性引物,建立了实时荧光定量PCR检测方法. 利用从污水中分离出的目的核酸片段构建重组质粒,通过测序和BLAST比对分析,确定了PCR扩增的特异性. 将重组质粒作为模板,分别测定标准曲线,在eaeA基因模板量8.77～8.77×105 copy,rfbE基因模板量4.98～4.98×105copy的范围内,Ct(循环阈值)与模板量的对数值具有良好的线性关系〔R2为0.997(eaeA)和1.000(rfbE)〕. 结合膜吸附洗脱浓缩方法,对于实际水样,eaeA和rfbE基因的检测限分别为1.75×102和9.96×101copy/100 mL. 利用该方法对城市污水处理厂进、出水进行检测的结果显示,污水二级处理工艺对这2种毒力基因的去除效果明显.
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| 关 键 词： | 病原性大肠埃希氏菌 eaeA rfbE 实时荧光定量PCR 城市污水 |
| 收稿时间： | 2011/11/29 0:00:00 |
| 修稿时间： | 2012/4/19 0:00:00 |
Quantitative Detection of Virulence Genes eaeAand rfbEof Pathogenic Escherichia coliin Municipal Wastewater by Real-Time PCR |
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| XIE Run-xin,ZHANG Chong-miao,WANG Xiao-chang,SUN Ting-ting and LI Xian.Quantitative Detection of Virulence Genes eaeAand rfbEof Pathogenic Escherichia coliin Municipal Wastewater by Real-Time PCR[J].Research of Environmental Sciences,2012,25(8):922-926. |
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| Authors: | XIE Run-xin ZHANG Chong-miao WANG Xiao-chang SUN Ting-ting and LI Xian |
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| Institution: | Key Laboratory of Northwest Water Resources, Ecology and Environment, Ministry of Education, School of Environmental and Municipal Engineering, Xi''an University of Architecture and Technology, Xi''an 710055, China;Key Laboratory of Northwest Water Resources, Ecology and Environment, Ministry of Education, School of Environmental and Municipal Engineering, Xi''an University of Architecture and Technology, Xi''an 710055, China;State Environmental Protection Key Laboratory of Microorganism Application and Risk Control MARC, Tsinghua University, Beijing 100084, ChinaAbstract:;Key Laboratory of Northwest Water Resources, Ecology and Environment, Ministry of Education, School of Environmental and Municipal Engineering, Xi''an University of Architecture and Technology, Xi''an 710055, China;Key Laboratory of Northwest Water Resources, Ecology and Environment, Ministry of Education, School of Environmental and Municipal Engineering, Xi''an University of Architecture and Technology, Xi''an 710055, China;Key Laboratory of Northwest Water Resources, Ecology and Environment, Ministry of Education, School of Environmental and Municipal Engineering, Xi''an University of Architecture and Technology, Xi''an 710055, China |
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| Abstract: | For the pathogenic bacteriaEscherichia coli, a quantitative detection method targeting the virulence genes eaeA and rfbE was established by real-time PCR technology. A recombinant plasmid was constructed by cloning purified DNA fragment from positive water sample into vector. By BLAST analysis, the specificity of amplification was identified. Utilizing the recombinant plasmid as the targeted gene standard, a good linear correlation between the Ct values and the logarithm of input copy numbers was found while the eaeAgene ranged from 8.77 to 8.77×105 copy (R2=0.997), and the rfbEgene ranged from 4.98 to 4.98×105copy (R2=1.000). Combined with the concentration procedure, the detection limit was 1.75×102 copy per 100 mL water sample for eaeA, and 9.96×101 copy/100 mL for rfbE. The results indicated that the municipal wastewater treatment process could effectively remove these virulence genes. |
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