应用实时荧光定量PCR定量检测溃疡性结肠炎肠道大肠埃希菌、乳酸杆菌及双歧杆菌属的变化

目的应用实时荧光定量PCR（RT—PCR）法对溃疡性结肠炎（ulcerative colitis，UC）患者粪便中大肠埃希菌、乳酸杆菌及双歧杆菌属的数量进行定量检测分析。方法根据细菌的16SrDNA基因序列设计大肠埃希菌、乳酸杆菌及双歧杆菌属的种属特异性引物。收集溃疡性结肠炎患者及正常对照者新鲜粪便标本各35例，从待测粪便标本中提取细菌基因组DNA，进行实时荧光定量PCR反应，定量分析不同细菌的数量。结果正常对照组与病例组粪便中细菌数量分别为大肠埃希菌（4．62±1．10；5．27±1．02）、乳酸杆菌属（4．99±0．75；4．65±0．95）、双歧杆菌属（5．07±0．95；4．93±0．99），病例组大肠埃希菌数量明显增多（t=2．540，P=0．013），而乳酸杆菌及双歧杆菌属数量与正常组比较差异无统计学意义（t1=0．488，P，=0．530；t2=-0．533，P：=0．596）。结论溃疡性结肠炎患者粪便中大肠埃希菌的数量较正常对照明显增多，而乳酸杆菌及双歧杆菌属的数量无明显变化，提示大肠埃希菌与溃疡性结肠炎的发病或复发有关系，而乳酸及双歧杆菌属与此病的关系有待进一步研究。

Application of fluorescent real-time polymerase chain reaction in quantification of Escherichia coli,Lactobacillus and Bifidobacteria in ulcerative colitis patients

Objective To detect the amount of Escherichia coli,Lactobacillus and Bifidobacteria in UC patients′ fecal specimens using fluorescent real-time PCR.Method Species or group-specific primers for Escherichia coli,Lactobacillus and Bifidobacteria groups were designed according to 16S rDNA sequence.Fresh fecal samples of UC patients(n=35) and healthy people(n=35) were collected;bacterial genome DNAs were extracted from them,then the extracted DNAs were quantified by fluorescent real-time PCR to analyze different bacterial amounts.Result The amounts of Escherichia coli,Lactobacillus and Bifidobacteria groups were respectively(4.62±1.10;5.27±1.02),(4.99±0.75;4.65±0.95),(5.07±0.95;4.93±0.99) in the fecal samples of the normal control group and the case group.The amount of Escherichia coli increased apparently in case group(t=2.540,P=0.013),while Lactobacillus and Bifidobacteria groups had no obvious difference from the normal group(t1=0.488,P1=0.530;t2=-0.533,P2=0.596).Conclusion Escherichia coli′s amounts obviously increase in UC patients′ fecal samples,while Lactobacillus and Bifidobacteria group′s amounts have no changes.It indicates that Escherichia coli is related to the incidence or recurrence of UC,and the relationship between Lactobacillus,Bifidobacteria groups and UC need be further studied.