采用16S rRNA高通量测序技术分析鲜奶中微生物的多样性  

Microbial diversity analysis of bovine fresh milk by high-throughput sequencing of metagenomic 16S rRNA

作　　者：吕成龙 田雨 陈芳慧 刘小军 李丽丽[4] 吕林雪 葛继文 顾晨浩 邹彦 王根林[1,2] 蔡亚非 LU Chenglong;TIAN Yu;CHEN Fanghui;LIU Xiaojun;LI Lili;L Linxue;GE Jiwen;GU Chenhao;ZOU Yan;WANG Genlin;CAI Yafei(College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,China;Dairy Herd Improvement Determination Center of Jiangsu Province,Nanjing 210095,China;Nanjing Weigang Dairy Co.,Ltd.Nanjing 211100,China;DHI Standard Material Lab,National Animal Husbandry Station,Beijing 100125,China)

机构地区：[1]南京农业大学动物科技学院,江苏南京210095 [2]江苏省奶牛生产性能测定中心,江苏南京210095 [3]南京卫岗乳业有限公司,江苏南京211100 [4]全国畜牧总站DHI标准物质实验室,北京100125

出　　处：《南京农业大学学报》2020年第2期333-338,共6页Journal of Nanjing Agricultural University

基　　金：江苏农业自主创新资金[SCX(18)3014];国家重点研发计划项目(2018YFC1200201);农业技术试验示范与服务支持项目(060-HY0045)

摘　　要：[目的]本研究以16S rRNA为分子标记,采用高通量测序技术分析探讨3种水平体细胞鲜奶样品中细菌种类、菌群多样性和菌群结构。[方法]选取江苏某牧场45头奶牛鲜奶样品,分为健康、亚临床乳腺炎型和临床乳腺炎型3组,并对临床型奶样进行细菌培养鉴定,同时分别提取各组鲜奶样品中细菌总DNA,根据16S rRNA V4—V5区设计引物进行扩增测序,利用生物信息学软件对测序结果进行分析。[结果]对体细胞数>1×106 mL-1的奶样进行细菌培养,鉴定出奶牛乳腺炎致病菌为无乳链球菌。对序列进行相似性聚类分析,得到394个可操作分类单位(OTU)。通过对OTU的聚类分析发现3组样品之间部分OUT水平存在明显差异。物种及丰度分析显示,门水平上的优势门为厚壁菌门(Firmicutes)、蓝藻门(Cyanobacteria)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobactice);属分类水平上的优势属为芽胞杆菌属(Bacillus)、乳球菌属(Lactococcus)、海洋杆菌属(Oceanobacillus)、沙雷菌属(Serratia)、鞘氨醇单胞菌属(Sphingomonas)和vadinBC27 wastewater-sludge group。其中临床组沙雷菌属和鞘氨醇单胞菌属显著高于其他2组,而海洋杆菌属的相对丰度降低。[结论]该牧场奶牛乳腺炎致病菌为无乳链球菌,为乳腺炎临床的抗生素选择和治疗提供了依据和借鉴。[Objectives]The objective of this study was to use the metagenomics pyrosequencing of bacterial 16S rRNA genes to investigate bacterial DNA diversity in milk samples of mastitic and healthy dairy cows and compare the results with those obtained by classical bacterial culture.[Methods]Forty-five milk samples were collected from a dairy cattle farm in Jiangsu,with fifteen of them showing signs of mastitis and used for microbiological culture.Bacterial genome DNA was extracted from milk and molecular segments of the V4-V5 region of the 16S rRNA gene was obtained by PCR,cloning and sequencing.[Results]Bacterial culture was carried out on milk samples with the number of somatic cells more than 1 million per milliliter,and the pathogenic bacteria of cow mastitis was identified as Streptococcus agalactiae.Identification of bacteria at different taxonomic levels was performed based on the 16S rDNA database.Microbial diversity in each groups was analyzed separately and compared with each other and we obtained 394 operational taxonomic units(OTU).Through the classification analysis of OTU,it was found that there were significant differences in partial OUT levels among the three groups of samples.In terms of species and abundance analysis,the dominant phylum in the phylum classification level was mainly Firmicutes,Cyanobacteria,Proteobacteria,Bacteroidetes and Actinobacteria,respectively.The dominant genera in the genus classifica-tion level were mainly Bacillus,Lactococcus,Oceanobacillus,Serratia,Sphingomonas and vadinBC27 wastewater-sludge group,among which Serratia and Sphingomonas in the clinical group were significantly higher than the other groups,while the relative abundance of Oceanobacillus decreased significantly.[Conclusions]The experiment found that the pathogenic bacteria of cow mastitis in this dairy farm was Streptococcus agalactiae,which provided a basis and reference for antibiotic selection and targeted treatment in dairy farm.

关 键 词：鲜奶 微生物多样性 菌群分布 16SrRNA 

分 类 号：S812.3[农业科学—草业科学]