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活性污泥DNA样品的保存条件对聚磷菌FQ-PCR结果的影响

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范念斯, 齐嵘, MINOTakashi, FUKUSHIMAToshikazu, 杨敏. 活性污泥DNA样品的保存条件对聚磷菌FQ-PCR结果的影响[J]. 环境工程学报, 2017, 11(3): 1362-1368. doi: 10.12030/j.cjee.201511020
引用本文: 范念斯, 齐嵘, MINOTakashi, FUKUSHIMAToshikazu, 杨敏. 活性污泥DNA样品的保存条件对聚磷菌FQ-PCR结果的影响[J]. 环境工程学报, 2017, 11(3): 1362-1368. doi: 10.12030/j.cjee.201511020
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FAN Niansi, QI Rong, MINO Takashi, FUKUSHIMA Toshikazu, YANG Min. Effects of DNA storage and quantification conditions of activated sludge sample on detecting polyphosphate accumulating organisms by FQ-PCR[J]. Chinese Journal of Environmental Engineering, 2017, 11(3): 1362-1368. doi: 10.12030/j.cjee.201511020
Citation: FAN Niansi, QI Rong, MINO Takashi, FUKUSHIMA Toshikazu, YANG Min. Effects of DNA storage and quantification conditions of activated sludge sample on detecting polyphosphate accumulating organisms by FQ-PCR[J]. Chinese Journal of Environmental Engineering, 2017, 11(3): 1362-1368. doi: 10.12030/j.cjee.201511020
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活性污泥DNA样品的保存条件对聚磷菌FQ-PCR结果的影响

  • 1.

    中国科学院生态环境研究中心, 环境水质学国家重点实验室, 北京 100085

  • 2.

    东京大学工学系研究科水环境技术研究中心, 柏市 113-8656

  • 基金项目:

    国家自然科学基金资助项目（51378489，31170468）

  • 中图分类号: X172

Effects of DNA storage and quantification conditions of activated sludge sample on detecting polyphosphate accumulating organisms by FQ-PCR

  • 1.

    State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

  • 2.

    Research Center for Water Environment Technology, Graduate School of Engineering, The University of Tokyo, Kashiwa 113-8656, Japan

  • Fund Project:

  • 摘要: 对目标微生物的准确定量是研究微生物群落的组成与功能的重要前提。荧光定量PCR技术作为目前流行的核酸定量分析技术，具有操作时间短，敏感性高，特异性强等特点，但也存在由客观因素导致实验结果出现显著误差的缺点。针对目前环境微生物定量方法中容易忽略的污泥样品DNA保存步骤与上机时间等环节，详细探讨了保存条件、上机批次对聚磷菌（PAOs）荧光定量PCR结果的影响。结果显示保存温度、保存时间均对标准质粒和污泥DNA样品中PAOs定量结果产生影响：模板DNA的浓度及保存时间会影响标准曲线Ct值的重复性；同一样品在相同保存温度（4℃）、不同保存时间、不同批次条件下的定量结果存在显著性差异（PPP>0.05）。因此，在对活性污泥样品进行定量分析时，DNA样品应平行保存于-20℃条件，尽量避免反复冻融，并确保对比的生物样品保存及上机条件完全一致，从而减少定量结果误差。
    Abstract: The precise quantification of the microorganisms is a basic requirement for the investigation of microbial community composition and function. Fluorescent quantitative PCR,as one of the most widely applied quantitative techniques,has several advantages like time saving and high sensitivity. However,errors caused by several objective factors can influence the quantitative results. In this case,the effects of hypothetic factors like storage conditions and operation batch order were investigated by phosphorus accumulating organisms (PAOs)for quantifying the raw activated sludge. The matrix results indicated that both the storage temperature and the storage period influenced the quantitative values of the sludge DNA samples significantly. Initial concentration of template DNA and the storage time affected the Ct value repeatability of standard curves,while different storing periods under relatively higher storage temperature (4℃)altered the qPCR values significantly (PPP>0.05) between two parallel samples stored at -20℃ for varying storage periods. Therefore,it is necessary to store the activated sludge sample for qPCR measurement in parallel,at -20℃ and to operate under the same batch order for subsequent horizontal comparison.
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  • 收稿日期:  2015-12-02
  • 刊出日期:  2017-03-10

活性污泥DNA样品的保存条件对聚磷菌FQ-PCR结果的影响

  • 1. 中国科学院生态环境研究中心, 环境水质学国家重点实验室, 北京 100085
  • 2. 东京大学工学系研究科水环境技术研究中心, 柏市 113-8656
  • 收稿日期:  2015-12-02
基金项目:

国家自然科学基金资助项目（51378489，31170468）

摘要: 对目标微生物的准确定量是研究微生物群落的组成与功能的重要前提。荧光定量PCR技术作为目前流行的核酸定量分析技术，具有操作时间短，敏感性高，特异性强等特点，但也存在由客观因素导致实验结果出现显著误差的缺点。针对目前环境微生物定量方法中容易忽略的污泥样品DNA保存步骤与上机时间等环节，详细探讨了保存条件、上机批次对聚磷菌（PAOs）荧光定量PCR结果的影响。结果显示保存温度、保存时间均对标准质粒和污泥DNA样品中PAOs定量结果产生影响：模板DNA的浓度及保存时间会影响标准曲线Ct值的重复性；同一样品在相同保存温度（4℃）、不同保存时间、不同批次条件下的定量结果存在显著性差异（PPP>0.05）。因此，在对活性污泥样品进行定量分析时，DNA样品应平行保存于-20℃条件，尽量避免反复冻融，并确保对比的生物样品保存及上机条件完全一致，从而减少定量结果误差。

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