摘要
背景:具核梭杆菌(Fn)是一种常见的肠道细菌,其可能作为条件致病菌参与了炎症性肠病(IBD)的发生。目前尚缺乏IBD患者肠道Fn的定量数据。目的:建立绝对定量real-time PCR方法,检测克罗恩病(CD)患者的粪便Fn,分析其与CD炎症指标的相关性。方法:收集苏州市立医院57例CD患者和41例健康体检者的粪便样本,提取基因组DNA。构建Fn菌株ATCC 25586特异的nusG(transcription antitermination protein)基因至pUC57质粒,作为标准品,以SYBR Green real-time PCR法检测梯度稀释的标准品质粒,建立绝对定量标准曲线。评价该检测方法的敏感性、特异性和稳定性,并以之检测CD患者和健康对照者的粪便Fn丰度,分析Fn丰度与粪便钙卫蛋白(FC)、C反应蛋白(CRP)和红细胞沉降率(ESR)的相关性。结果:本研究建立的粪便Fn绝对定量real-time PCR方法敏感性高、特异性强、稳定性好。CD患者粪便Fn检出率和丰度均显著高于健康对照者(P=0.034;P=0.039),且Fn丰度与FC水平呈显著正相关(r=0.459,P=0.011)。结论:本研究建立的绝对定量real-time PCR方法可用于人粪便样本Fn丰度检测。苏州地区CD患者粪便Fn检出率和丰度显著升高,Fn丰度与炎症活动度指标FC相关。
Background:Fusobacterium nucleatum(Fn)is a common resident of the human GI tract and has been recognized as an opportunistic pathogen implicated in inflammatory bowel disease(IBD).Few studies had focused on the quantitative analysis of Fn in IBD patients.Aims:To establish a novel absolute quantitative real-time PCR method for detection of Fn in fecal samples of Crohn’s disease(CD)patients and to study the correlation between fecal Fn and the common inflammatory indicators of CD.Methods:Fecal samples of 57 CD patients and 41 healthy subjects from Suzhou Municipal Hospital were collected and the genomic DNA was extracted.NusG(transcription antitermination protein)of Fusobacterium nucleatum subsp.nucleatum ATCC 25586 was constructed into pUC57 to form a standard plasmid.An absolute quantitative standard curve was built by detecting the gradient diluted standard plasmid via SYBR Green real-time PCR method.The sensitivity,specificity and stability of this novel method were assessed,and then the novel method was used to detect the abundance of Fn in fecal sample of CD patients and healthy controls.Correlations between fecal Fn and fecal calprotectin(FC),C-reactive protein(CRP)and erythrocyte sedimentation rate(ESR)were analyzed.Results:The absolute quantitative real-time PCR method for the detection of fecal Fn established in this study was sensitive,specific and stable.The detection rate and mean abundance of fecal Fn were significantly higher in CD patients than in healthy controls(P=0.034;P=0.039).Fecal Fn abundance in CD patients was positively correlated with FC level(r=0.459,P=0.011).Conclusions:The absolute quantitative real-time PCR established in this study is a promising method for human fecal Fn detection.In Suzhou,Jiangsu Province,the detection rate and abundance of fecal Fn are significantly increased in CD patients.Fecal Fn abundance in CD patients is correlated with FC level,and may reflect the disease activity.
作者
邢晔陈
胡彤
徐丽娟
张丽平
叶玉兰
庞智
尹娟
XING Yechen;HU Tong;XU Lijuan;ZHANG Liping;YE Yulan;PANG Zhi;YIN Juan(Department of Gastroenterology,the Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou,Jiangsu Province 215008;Digestive Disease and Nutrition Research Center,the Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou,Jiangsu Province 215008)
出处
《胃肠病学》
2020年第6期326-331,共6页
Chinese Journal of Gastroenterology
基金
江苏省基础研究计划(自然科学基金)面上研究项目(BK20161232)
南京医科大学科技发展基金(NMUB2018215)
苏州市临床重点病种诊疗技术专项(LCZX201715)。
关键词
CROHN病
具核梭杆菌
粪便钙卫蛋白
实时聚合酶链反应
Crohn Disease
Fusobacterium nucleatum
Fecal Calprotectin
Real-Time Polymerase Chain Reaction
