活性污泥中氨氧化菌实时荧光定量PCR标准品制备方法的比较

摘要

为了快速准确地检测活性污泥中氨氧化菌(AOB)的拷贝数,探求一种新的快速简便的绝对定量标准品的制备方法.利用纯化的PCR产物和重组质粒标准品分别进行绝对定量,比较两者熔解曲线、扩增曲线、标准曲线和绝对拷贝数等方面的差异,分析两种标准品制备方法应用方面的优势.结果显示,两种标准品制备的熔解曲线都呈单一尖峰、扩增曲线指数期平行、标准曲线R2＞ 99％,PCR产物作为标准品得到的AOB绝对拷贝数小于质粒标准品的结果(P＜0.05),表明纯化的PCR产物作为标准品较制备重组质粒标准品而言,操作过程更加简便、经济、对人员专业要求更低,在其纯度较高的情况下,具有和重组质粒标准品一样的应用效果.%To detect the copy number of ammonia oxidizing bacteria (AOB) of activated sludge rapidly and accurately,and to explore an easy preparation method of absolute quantitative PCR standard,this study used purified PCR product and preparative recombinant plasmid,respectively,to conduct absolute quantification PCR (AQ-PCR).The melting curve,amplification curve,standard curve and differences of absolute copy number were compared,the advantages of the two standard preparation method were analyzed.The results showed that the two melting curves of preparation standard were a single peak,the index phases of the amplification curves were parallel,R2 values of standard curves were more than 99％,the AOB absolute copy number of PCR product as a standard was less than the results of recombinant plasmid standard (P ＜ 0.05).It indicates the purification of PCR products as a standard is more convenient,more economic,and lower personnel professional requirements than the recombinant plasmid standard.In the case of the high purity,PCR product has the same application effect with recombinant plasmid standard.