中文摘要：

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（P〈0.05）;相同样品在不同保存温度、不同保存时间、同一批次条件下定量结果存在显著差异（P〈0.05）,平行保存于-20℃的污泥DNA样品不同保存时间、同一批次定量结果没有显著差异（P〉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。

英文摘要：

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 q PCR values significantly（ P〈0. 05）. Various quantitative results（ P〈0. 05） of the same sample were observed under different storage conditions（ temperature and time） within the same batch. However,there was no significant difference（ P〉0. 05） between two parallel samples stored at- 20 ℃ for varying storage periods. Therefore,it is necessary to store the activated sludge sample for q PCR measurement in parallel,at- 20 ℃ and to operate under the same batch order for subsequent horizontal comparison.