| 基于RT-qPCR选择性检测水中活性病原菌 |
| 摘要点击 1656 全文点击 922 投稿时间：2012-02-14 修订日期：2012-04-21 |
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| 中文关键词 逆转录定量PCR RNA 大肠杆菌 粪肠球菌 活性菌 |
| 英文关键词 reverse transcription q quantitative PCR (RT-qPCR) RNA E. coli Enterococcus faecium viable bacteria |
| 作者 | 单位 | E-mail | | 林怡雯 | 清华大学环境学院,环境模拟与污染控制国家重点联合实验室,北京 100084 | lyw10@mails.tsinghua.edu.cn | | 李丹 | 清华大学环境学院,环境模拟与污染控制国家重点联合实验室,北京 100084 | | | 吴舒旭 | 清华大学环境学院,环境模拟与污染控制国家重点联合实验室,北京 100084 | | | 何苗 | 清华大学环境学院,环境模拟与污染控制国家重点联合实验室,北京 100084 | hemiao@mail.tsinghua.edu.cn | | 杨天 | 清华大学环境学院,环境模拟与污染控制国家重点联合实验室,北京 100084 | |
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| 中文摘要 |
| 以大肠杆菌和粪肠球菌作为研究对象,研究建立了一种逆转录定量PCR(reverse transcription q quantitative PCR,RT-qPCR)方法,以选择性检测水中活性病原菌. 研究结果表明,细菌体内的RNA经过RT-PCR逆转录成cDNA后利用qPCR可定量目的基因拷贝数,对处于稳定生长期的大肠杆菌(培养6~18 h)和粪肠球菌(培养10~38 h)体内的RNA含量进行检测分别为1 copies·CFU-1和7.98×102 copies·CFU-1,以此作为细菌定量的依据,达到准确定量检测水体中目的基因RNA拷贝数而确定活性细菌含量的目的. 通过3种方法(培养法、qPCR、RT-qPCR)检测热灭活后的大肠杆菌和粪肠球菌,结果表明与qPCR相比,RT-qPCR能够区分1.43 lg copy(大肠杆菌)与2.5 lg copy(粪肠球菌)的非活性菌,进而选择性检测活性病原菌. 实际水样底物基质对RT-qPCR方法的影响不大,RT-qPCR与培养法之间有较好的线性相关性(大肠杆菌, R2=0.930,粪肠球菌,R2=0.948),本研究建立的方法可以用于实际水样活性病原菌的检测. |
| 英文摘要 |
| A reverse transcription q quantitative PCR (RT-qPCR) assay method was established, which can quantify the copy numbers of RNA in pathogenic bacteria of E. coli and Enterococcus faecium. The results showed that cDNA was generated with the RT-PCR reagents, target gene was quantified with the qPCR, the copy numbers of RNA were stable at about 1 copies·CFU-1 for E. coli and 7.98×102 copies·CFU-1 for Enterococcus faecium respectively during the stationary grow phase for the both indicator bacteria ［E. coli (6-18 h) and Enterococcus faecium(10-38 h)］. The established RT-qPCR method can quantify the numbers of viable bacteria through detecting bacterial RNA targets. Through detecting the heat-treated E. coli and Enterococcus faecium by three methods (culture method, qPCR, RT-qPCR), we found that the qPCR and RT-qPCR can distinguish 1.43 lg copy non-viable E. coli and 2.5 lg copy non-viable Enterococcus faecium. These results indicated that the established methods could effectively distinguish viable bacteria from non-viable bacteria. Finally we used this method to evaluate the real effluents of the secondary sedimentation of wastewater treatment plant (WWTP), the results showed that the correlation coefficients (R2) between RT-qPCR and culture method were 0.930 (E. coli) and 0.948 (Enterococcus faecium), and this established RT-PCR method can rapidly detect viable pathogenic bacteria in genuine waters. |
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