细菌16S rDNA荧光定量PCR法分析溃疡性结肠炎患者肠道菌群变化

16S rDNA-based fluorescent real-time PCR analysis of the variation of fecal microbiota in patients with ulcerative colitis

目的应用实时荧光定量PCR技术对溃疡性结肠炎(ulcerative colitis,UC)患者的粪便菌群进行定量分析。方法研究对象包括30名UC活动期、30名UC缓解期患者及22名正常对照,收集其粪便标本。根据细菌的16S rDNA序列设计双歧杆菌属、乳酸杆菌属、肠球菌属和大肠杆菌的特异性引物。待测粪便标本提取细菌基因组DNA,进行实时荧光定量PCR反应测定16S rDNA拷贝数,分析不同细菌的数量。结果UC活动期患者与正常对照相比,粪便中双歧杆菌(8.45±0.92vs9.10±0.78)、乳酸杆菌(8.11±0.94vs8.69±0.55)数量明显减少(P<0.05),大肠杆菌(9.88±1.34vs9.71±0.92)和肠球菌(7.74±0.63vs7.61±0.49)无明显变化(P>0.05);而UC缓解期患者粪便中4种细菌数量(双歧杆菌9.15±0.81,乳酸杆菌8.51±0.80,大肠杆菌9.54±1.64,肠球菌7.90±0.46)均与正常对照无明显差异(P>0.05)。结论UC活动期患者粪便双歧杆菌、乳酸杆菌数量较正常对照明显减少,而UC缓解期患者粪便菌群与正常对照无明显差异。提示肠道菌群失调可能是具有UC遗传易感性个体发病的触发因素。

Objective To analyze the fecal microbiota of UC patients subjects using fluorescent real-time PCR. Methods Fresh fecal samples were obtained from patients with active UC （ n = 30） , UC in remission （ n = 30） , and healthy volunteers （n = 22）. A set of 16S rDNA-targeted group-or species-specific primers for Bifidobacterium spp. , Lactobacillus group, Enterococcus spp. and Escherichia coli were designed. 16S rDNA copy numbers of bacterial genome DNA extracted from fecal samples were quantified by real-time PCR to analyze bacterial amounts. Results The levels of Bifidobacterium spp. （8.45 ＋0.92 vs9. 10 ＋0.78） and Lactobacillus group （8. 11 ＋0.94 vs 8.69 ＋0.55） in active UC samples were significantly lower than those of healthy controls （P 〈 0.05 ） , whereas Enterococcus spp. （7.74 ＋ 0.63 vs 7.61 ＋0.49） and Escherichia coli （9.88 ＋ 1.34 vs 9.71 ＋0.92） levels did not show significant difference （P 〉 0.05）. However, all four fecal bacteria quantities of UC patients in remission （9. 15 ＋0.81, 8.51 ＋0.80, 7.90 ＋ 0.46, 9.54 ＋ 1.64, respectively） were similar to those of healthy controls （P 〉 0.05 ）. Conclusion The fecal flora composition of active UC patients had a significaut deerease in Bifidobacterium spp. and Lactobacillus group, whereas UC patients in remission had no differences with those of healthy controls. These data underline the central role of intestinal microbiota dysbiosis in UC, which might be a trigger of the intestinal inflammation in individuals with genetic susceptibility.