肠道常见菌群荧光定量PCR检测方法的建立  

Establishment of real-time PCR method for detection of intestinal bacteria

作　　者：程悦[1] 左浩江[1] 廖虹瑜[2] 陈嘉熠[1] 郑田利[1] 裴晓方[1] 许欣[1] 

机构地区：[1]四川大学华西公共卫生学院(华西第四医院),四川成都610041 [2]四川省疾病预防控制中心,四川成都610041

出　　处：《现代预防医学》2014年第23期4338-4341,4359,共5页Modern Preventive Medicine

基　　金：粗壮女贞提取物对肠道菌群的调节与减肥机制的研究(国家自然科学基金项目81273055)

摘　　要：目的分别建立检测部分肠道常见菌群的荧光定量PCR方法,为肠道菌群及其相关疾病的研究提供快速、敏感、准确的检测手段。方法根据肠道细菌16S r RNA基因序列,选择针对不同肠道细菌的特异性引物,建立并优化荧光定量PCR检测方法,并对建立的方法进行特异性及重复性实验。采用基因重组技术,构建肠道常见菌群目的基因的重组质粒,作为PCR检测定量参考。结果成功建立了检测大肠埃希菌、肠球菌、乳杆菌、双歧杆菌、产气荚膜梭菌、拟杆菌、拟杆菌门菌、硬壁菌门菌以及肠道菌群总量的荧光定量PCR方法。方法特异性试验结果表明,反应体系与待测菌以外的其他细菌无交叉反应。重复试验结果显示,各待测菌8次重复试验的变异系数均小于2%。各待测菌标准曲线的相关系数r≥0.994,线性关系良好。结论本研究建立的Eva Green荧光定量PCR检测方法可以特异的从粪便中检出部分肠道常见菌群,且方法重复性好,是快速、敏感、准确地检测肠道菌群的有利手段。Objective To establish a real-time PCR method for detection of 9 intestinal bacterium, provide a rapid, specific and sensitive tool for research of intestinal bacteria and the related metabolic diseases. Methods Primers were designed for every intestinal bacteria based on the sequences of 16 s RNA. Established PCR method, optimized reaction conditions. Then its specificity and repeatability were investigated. Recombinant plasmid were constructed by genetic recombination technology as the reference of quantitative detection. Results The real-time PCR method for detection of Escherichia coli, Enterococcus, Bifidobacterium,Lactobacillus, Clostridium perfringens, Bacteroides, Bacteroidetes, Firmicutes and the all intestinal bacteria were established. The trails of specificity showed there was no cross reaction among these systems. The repeated trials showed the coefficients of variation were all below 2%. Standard curve showed a good linear relation, r≥0.994. Conclusion The established real-time PCR method could detect some intestinal bacterium specifically from feces with high repeatability, which provides a rapid, specific and sensitive tool for research of intestinal bacteria.

关 键 词：荧光定量PCR 肠道菌群 重组质粒标准品 

分 类 号：R115[医药卫生—公共卫生与预防医学]