PCR-DGGE 技术应用于手足口病儿童口腔微生物菌群变化的初步研究

摘要

Objective To apply PCR-DGGE technology analysing changes of oral microbial floras in children with hand foot and mouth disease( HFMD) .Methods According to the random number table method,3 children with HFMD were randomly chosen and their saliva samples were collected;at the same time, saliva samples were collected from one normal child,and from one child with herpetic ginggivostomatitis(HGS).Samples′total DNA extraction of the microbial community were performed;taking it as template for amplification of 16 s rRNA V3 variable area, the structure of product by the DGGE fingerprint, and using UVIBAND/MAP software for comparing similarity index of the community fingerprint;the edge banding on swimming belt of DGGE fingerprint were excised from the gel for se-quencing, gene sequence analysis was performed, then the sample advantage bacterium group was obtained.Results The advantage bacterial colonies of HFMD group:Capnocytophaga, Selenomonas, Prevotella, TM7_genera_incertae_sedis, Streptococcus, Neisseria, Porphyromonas, Campylobacter, Campylobacter, Granulicatell-a, Leptotrichia, Veil-lonella, Bacteroides-like sp, Actinomyces, Fusobacterium.The advantage bacterial colonies of normal group: Veil-lonella, Capnocytophaga, Prevotella,Gemella , Porphyromonas, Ruminococcaceae, Lachnospiraceae, Selenomonas, Clostridium, Haemophilus.The advantage bacterial colonies of HGS group: Lactococcus,Prevotella.Conclusion The flora structure of HFMD group was more complex, and there was obvious difference in the flora structure between HFMD group and HGS group.%目的：应用PCR-DGGE技术对手足口病（ HFMD）、疱疹性口腔炎（ HGS）及正常儿童口腔微生物菌群变化进行初步分析，以期了解三者是否存在差异。方法按随机数字表法选择3例手足口病唾液样本，1例幼儿园正常儿童唾液以及1例疱疹性口腔炎患儿唾液。样本进行微生物群落总DNA的抽提；以此为模板扩增16S rRNA V3可变区，产物经DGGE指纹图谱分析其组成结构，并运用UVIBAND／MAP等软件比较所得群落指纹图谱的相似性指数；对DGGE指纹图谱上的泳带中优势条带切胶测序，进行基因序列分析，得出该样本的优势菌群。结果手足口病组优势菌群：Capnocytophaga嗜二氧化碳噬细胞菌、Selenomonas月形单胞菌属、Prevotella普氏菌属、TM7＿genera＿incertae＿sedis菌属、Streptococcus链球菌属、Neisseria奈瑟菌属、Porphyromonas卟啉单胞菌属、Campylobacter弯曲菌属、Campylobacter弯曲菌属、Granulicatella菌属、Leptotrichia纤毛菌属、Veillonella 韦永氏球菌属、Bacteroides-like sp菌属、Actinomyces放线菌、Fusobacterium梭菌属。正常对照组优势菌群：Veillonella韦永氏球菌属、Capnocytophaga嗜二氧化碳噬细胞菌、 Prevotella普氏菌、Gemella双胎溶血菌属、Porphyromonas卟啉单胞菌属、Ruminococcaceae瘤胃球菌属、Lachnospiraceae毛螺菌属、Selenom-onas月形单胞菌属、Clostridium梭状芽孢杆菌、Haemophilus嗜血杆菌。疱疹性口腔炎组优势菌群：Lactococcus乳球菌属、Prevotella普氏菌属。结论手足口病组菌群结构更为复杂，与疱疹性口腔炎组相比菌群结构差异明显。