本研究旨在建立对肠道主要共生菌的快速定量方法。根据细菌16S rRNA基因的保守序列并参考相关研究，设计针对总肠道菌群的通用引物和探针，以及针对双歧杆菌属、肠球菌属和肠杆菌科的特异性引物和探针，采用引物设计工具(Primer-BLAST) 以及聚合酶链式反应(polymerase chain reaction, PCR)扩增以验证引物和探针的特异性。通过分子克隆技术，构建各目标菌属目的基因的重组质粒作为qPCR检测的标准模板，选择标准模板进行重复性实验，并计算组内和组间重复变异系数。用所建立的方法对不同年龄段的临床粪便标本进行3类细菌的检测，并初步分析这些菌群与增龄的关系。结果显示，引物和探针具有较高的特异性；各目的细菌标准曲线在一定范围内线性关系良好，总菌群或各目标菌属的线性范围分别为：总菌群2.9×10³～2.9×10¹²copies/μL、双歧杆菌3.1×10²～3.1×10⁹ copies/μL、肠球菌5.9×10²～5.9×10⁹ copies/μL、肠杆菌6.3×10²～6.3×10⁹ copies/μL，决定系数R²≥ 0.995；检测的最低拷贝数为总菌群7.5×10² copies/μL、双歧杆菌 46 copies/μL、肠球菌 37 copies/μL、肠杆菌 51 copies/μL；重复性实验变异系数在1.32%以下，具有良好的可重复性。对不同年龄段临床粪便标本检测的结果显示，肠球菌和肠杆菌含量随增龄呈增长趋势，且老年组含量显著高于青年组，但在高龄老年人中未发现肠球菌数量增加。双歧杆菌含量随增龄减少，且各组间差异显著，在高龄老年人中也未观察到双歧杆菌显著减少。结果提示，本研究建立了一种可供临床推广的菌群绝对定量方法，能够同时检测3类细菌和菌群总量，对于菌群定量研究具有实际意义。

We established a rapid quantification method for detecting the absolute amount of total bacteria, Bifidobacterium, Enterococcus, and Enterobacteriaceae in gut microbiota. The recombinant plasmids for the target DNA fragments of each target bacterial taxa were constructed by molecular clone technique and used as a standard template for quantitative assays. The results showed the primers and probes were highly specific. The linearity of different bacterial standard curves had a high coefficient of determination (R²≥0.995). The linear range was 2.9×10³-2.9×10¹² copies/μL for all bacteria, 3.1×10²-3.1×10⁹ copies/μL for Bifidobacterium, 5.9×10²-5.9×10⁹ copies/μL for Enterococcus and 6.3×10²-6.3×10⁹ copies/μL for Enterobacteriaceae. The low threshold of copy number was 7.5×10²copies/μL for total bacteria, 46 copies/μL for Bifidobacterium, 37 copies/μL for Enterococcus, 51 copies/μL for Enterobacteriaceae, respectively. Moreover, the detection limit was 10 times higher than that of qPCR dye method. Besides, the coefficient of variation of the reproducibility experiment was below 1.32%，implying a well reproducibility. The method was used to detect clinical fecal samples of different ages, and the relationship between these bacteria account and ageing was analyzed, showing high consistent with those reported in other researches. In conclusion, a clinically applicable method for quantification of microbiota was established, which can detect 3 types of bacteria and total bacteria at the same time. And, it has some positive significance for the study of microbiome.