sybrgreenⅰ实时荧光定量pcr法分析肠应激综合征儿童肠道菌群.doc

SYBR GreenⅠ实时荧光定量PCR法分析肠应激综合征 儿童肠道菌群变化 华川1，曹海涛2*（解放军252医院：1.检验科；2.医务处，河北保定 071000） 摘要 目的 应用SYBR Green Ⅰ实时荧光定量PCR法分析1-6岁肠应激综合征儿童患者肠道菌群的变化，探讨肠道相关分子微生态在发病中的作用及意义。方法 根据16SrRNA基因序列设计双歧杆菌属、乳酸杆菌属、肠球菌属、大肠杆菌、拟杆菌属、梭杆菌属、梭菌属的特异性引物，收集25份患者粪便标本及25份正常对照标本，提取细菌基因组DNA，应用SYBR Green Ⅰ实时荧光定量PCR反应测定7种细菌的数量。 结果 正常对照组与肠应激综合征儿童粪便中细菌数量为双歧杆菌属：8.85±0.57比8.28±0.68，乳酸杆菌属：8.36±0.45比7.79±0.39，梭杆菌属：8.12±0.44比6.07±0.46，梭菌属：7.32±0.63比6.66±0.65，发病组的数量明显减少（P 0.05），大肠杆菌细菌数量的对数值分别为8.41±0.38比8.40±0.50，肠球菌数量的比值为7.49±0.65比7.96±0.42，拟杆菌属数量比值为10.62±0.47比10.64±0.55，2组数据没有明显差异。结论 肠道菌群的变化与儿童肠应激综合征的发生及发展机理有一定的关系,SYBR GreenI实时荧光定量PCR技术为儿童肠道菌群组成、动态变化及相关的研究提供了一种有效监测手段。 关键词： 肠应激综合征 肠道菌群 荧光定量 儿童 中图分类号：R725 文献标识码： A Analysis of the changes of intestinal microflora in patients with diarrhea in children by real time fluorescent quantitative PCR SYBR Green I Hua Chuan1,Cao Haitao2* (1.Department of Clinical Laboratory;2. Department of Medicine; No. 252 Hospital of PLA, Baoding 071000, China) Abstract objective: Analysis of the changes of 1-6 years old children with diarrhea intestinal flora in patients with application of SYBR Green real-time fluorescence quantitative PCR，To investigate the gut associated molecular micro ecological role in the pathogenesis and significance。Methods According to the specific primers of 16SrRNA gene sequence of Bifidobacterium, Lactobacillus, Enterococcus and Escherichia coli，Bacteroides, Fusobacterium, Clostridium. Collect 25 stool specimens and 25 normal control specimens，Extraction of bacterial genomic DNA，SYBR determination of real-time fluorescent quantitative PCR Green Ⅰ response quantity 7 kinds of bacteria. Results The normal control group and the number of bacteria in the stool diarrhea children for Bifidobacterium spp.: 8.85?.57 vs 8.28?.68, lactic acid bacteria of the genus: 8.36?.45 vs 7.79?.39, Fusobacterium: 8.12 ?0.44 vs 6.07 ?0.46 ,Clostridium: 7.32 ?0.63 vs 6.66 ?0.65significantly reduced the number of disease groups (P 0.05), Escherichia coli bacteria n