Establishment of an absolute quantitative method for special intestinal bacterial flora by TaqMan probe multiplex real-time fluorescent PCR
WANG Hong1, SHI Meifang2, YANG Hao1, WU Yonglin1, XIAO Zhenming1, Qiao Peng1, QU Yifan1, RONG Xingyu1, ZHAO Chao1，3
1. Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China; 2. Community Health Service Center of Youyi Community, Baoshan District, Shanghai 201999, China; 3. National Clinical Research Center for Aging and Medicine (Huashan Hospital Affiliated to Fudan University), Shanghai 200040, China
Abstract：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.
王虹1，施美芳2，杨浩1，邬永琳1，肖振明1，谯鹏1，屈一帆1，荣星喻1，赵超1，3. 基于TaqMan探针多重实时荧光PCR的肠道特殊菌群绝对定量方法的建立[J]. 微生物与感染, 2022, 17(3): 129-138.
WANG Hong1, SHI Meifang2, YANG Hao1, WU Yonglin1, XIAO Zhenming1, Qiao Peng1, QU Yifan1, RONG Xingyu1, ZHAO Chao1，3. Establishment of an absolute quantitative method for special intestinal bacterial flora by TaqMan probe multiplex real-time fluorescent PCR. JOURNAL OF MICROBES AND INFECTIONS, 2022, 17(3): 129-138.
2022, Vol. 17 
