基于高通量测序的营养不良儿童肠道菌群多样性研究

Intestinal flora diversity of malnourished children based on high-throughput sequencing

目的 使用高通量测序模式和方法 ,对比分析营养不良儿童和正常儿童中肠道菌群物种存在的多样性和实际组成特点存在的差异。方法 选取20例营养不良患儿作为A组,选择20例身体正常的儿童作为B组,采集分析粪便样本,提取细菌中的核糖体DNA(RDNA),通过两组聚合酶链式反应(PCR)模式和方法 ,增加V4~V5区域实现高通量的测序设计,并进行生物信息学的分析研究;构建相应的稀释曲线,使用MOTHUR软件实现APLHA的多样化的计算分析,通过R语言实现热图(HEAT MAP)分析研究,实现典型关联分析(CCA),之后分别在样本的门和属两个列表中实现数据的整合和归纳,进行统计分析。结果 两组年龄、性别、分娩方式等一般资料比较,差异无统计学意义(P>0.05)。两组生物多样性比较差异无统计学意义(P>0.05)。两组生物多样性比较差异无统计学意义(P>0.05)。两组样本中共发现微生物154种。A组肠道菌群的操作性分类单元(OTU)为320, B组大肠产甲烷菌群的OTU为348,以及其共同OTU为290,两组肠细菌群门的细菌中共检出14个。为更好地展现检测结果 ,本研究依托柱状图优势,将各个样品进行分类学层面的比较和研究。A组硬壁菌门及未分级菌(乳酸菌)的OTU少于B组,拟杆菌(多形状杆菌、罗斯菌)的OTU多于B组,差异有统计学意义(P<0.05)。除A组含有的菌群外, B组还独有厌氧弧菌属、厌氧螺菌属等19种,远超A组体内的菌群种类和丰度。两组体内多形杆状菌、隐秘杆菌属等5种菌群丰度相同,差异无统计学意义(P>0.05),其他菌群丰度比较差异有统计学意义(P<0.05)。结论 营养不良儿童肠道菌群中硬壁菌的数量较少,其中多形杆状菌和罗斯菌的浓度较高,为保持菌群的稳定性与均衡,必须利用不同菌属的实际特性做好菌落的调节,确保儿童有良好的消化吸收与身体生长发育。

Objective To compare and analyze the diversity and actual composition characteristics of proposed flora species between malnourished children and normal children by using high-throughput sequencing mode and method.Methods 20 malnourished children were selected as group A,and 20 children with normal health were selected as the control group.Stool samples were collected for analysis and discrimination,and ribosomal DNA(RDNA)in bacteria was extracted.Through two groups of polymerase chain reaction(PCR)models and methods,high-throughput sequencing design was achieved by increasing V4-V5 regions.In addition,the bioinformatics analysis and research were carried out,the corresponding dilution curve was constructed,the MOTHUR software was used to realize the diversified calculation and analysis of APLHA,the HEAT MAP analysis and research were realized through R language,and the canonical correspondence analysis(CCA)was realized.After that,the data were integrated and summarized in the two lists of sample gate and genus,respectively,for statistical analysis.Results There was no statistically significant difference between the two groups in the basic information of age,gender,mode of delivery(P>0.05).There was no statistically significant difference in biodiversity between the two groups(P>0.05).A total of 154 microbial species were found in the two groups of samples.The operational taxonomic unit(OTU)of intestinal flora of group A was 320,the OTU of group B colorectal methanogens was 348,and their common OTU was 290.A total of 14 species of intestinal flora were detected in the two groups of children.In order to better display the test results,this study relied on the advantages of bar charts to compare and study each sample at the taxonomic level.OTU of Firmicutes and ungraded bacteria(lactic acid bacteria)in group A was less than that in group B,and OTU of Bacteroides(Bacteroides thetaiotaomicron and Rothia)was more than that in group B.The difference was statistically significant(P<0.05).In addition to the flora contained in group A,group B also had 19 unique species such as Anaerovibrio and Anaerobiospirillum,which far exceeded the species and abundance of flora in group A.The abundance of 5 bacterial communities in the two groups was the same,including Bacteroides thetaiotaomicron and Arcanobacterium,but the difference was not statistically significant(P>0.05).The abundance of other bacterial communities showed statistical significance(P<0.05).Conclusion The number of Firmicutes in the intestinal flora of malnourished children is low,and the content of Bacteroides thetaiotaomicron and Rothia is high.Therefore,in order to ensure the stability and balance of the flora,it is necessary to adjust the colony by taking advantage of the actual characteristics of different bacteria genera to ensure that children have good absorption and physical development.