基于16S rRNA测序分析巴马小型猪性别与肠道微生物的关系

为了研究性别对巴马小型猪粪便微生物组成及丰度的影响,试验以10头雄性和10头雌性试验用巴马小型猪作为试验对象,收集新鲜粪便,采用16S rRNA测序技术和生物信息学分析方法,分析微生物多样性、组成并对预测的代谢通路进行了差异分析。结果表明：雄性巴马小型猪和雌性巴马小型猪之间微生物的Alpha多样性无显著差异(P>0.05)。在门水平上雄性和雌性巴马小型猪的核心菌群均为厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes);在属水平上雄性的核心菌群为志贺氏杆菌属(Shigella,5.81%)、颤螺菌属(Oscillospira,4.62%)、密螺旋体属(Treponema,4.14%)、梭菌属(Clostridiaceae＿Clostridium,2.73%)及拟杆菌属(Bacteroides,2.58%),雌性为颤螺菌属(5.23%)、密螺旋体属(4.94%)、链球菌属(Streptococcus,3.74%)、SMB53(2.35%)及梭菌属(2.12%)。在门水平上,雌性疣微菌门(Verrucomicrobia)的丰度显著高于雄性(P<0.05);在...     

正常和腹泻水貂肠道微生物16S rRNA测序差异性分析

为调查正常水貂和腹泻水貂肠道菌群差异及发病原因,本试验采集了正常和腹泻水貂的粪便进行16S rRNA测序,并展开微生物物种的分布和丰度聚类差异分析。结果显示,与正常水貂相比,腹泻水貂中厚壁菌门所占比例显著增多(P<0.05),变形菌门和丙型变形菌纲所占比例显著减少(P<0.05);梭菌纲、梭菌目、巴氏杆菌目、巴氏杆菌科、链球菌科、梭菌科、巴氏杆菌属、梭菌属、链球菌属、放线杆菌属所占比例均极显著增多(P<0.01),肠杆菌目、肠杆菌科、肠球菌科、沙雷氏菌属、肠球菌属所占比例均极显著减少(P<0.01)。由此可知,水貂肠道微生物中巴氏杆菌属、梭菌属、放线菌属、链球菌属等多种致病菌群占比及相对丰度显著增加是导致水貂腹泻的原因之一。本试验通过分析得出正常和腹泻水貂肠道内菌种的差异,对疾病的诊断和治疗有一定的意义,并对微生态制剂的发展提供了数据支持。     

基于16S rRNA高通量测序技术分析生鲜水牛乳在加工前的细菌多样性

本研究利用16S rRNA高通量测序分析生鲜水牛乳加工前的细菌多样性.分别提取刚挤出30min内,及冷藏5h、12h及24h的水牛乳样本细菌总DNA,PCR扩增其16S rDNA,利用纯化后的扩增片段构建其菌群的16S rDNA文库,采用Miseq PE300进行高通量测序及BLAST比对.结果显示,在属水平,刚挤出3...     

基于16 S rRNA测序分析冰鲜鸡腐败过程中微生物变化

为分析冰鲜鸡腐败过程中微生物变化,筛选冰鲜鸡腐败相关菌群,研究利用16 S rRNA测序技术分析了保存1、3、5和7d冰鲜鸡样本微生物组成和多样性,利用正交偏最小二乘法回归和逐步多元回归分析筛选与冰鲜鸡腐败相关的菌属.结果 显示:冰鲜鸡腐败过程中微生物丰富度和均匀度均显著下降,保存5d和7d样本与保存1d和3d样本微生...     

基于16S rRNA测序揭示杜洛克猪早期体重与肠道微生物的关系

本研究旨在探讨肠道微生物与杜洛克猪早期体重的关系。从91头80日龄杜洛克公猪中根据个体体重排序选择体重较轻(lighter body weight,LBW)的9头猪和体重较重(heavier body weight,HBW)的9头猪。通过对16S rRNA基因V3-V4区测序,分析了HBW和LBW组猪粪便中微生物多样性、组成和预测功能。结果显示,HBW和LBW组之间微生物的Alpha多样性无显著差异(P>0.05)。杜洛克仔猪肠道微生物的核心菌门为厚壁菌门(HBW和LBW组分别为65.62%和66.57%)和拟杆菌门(HBW和LBW组分别为30.87%和29.80%),核心菌属为普雷沃菌属(HBW和LBW组分别为23.82%和20.84%)、乳杆菌属(HBW和LBW组分别为9.11%和16.55%)、未分类的瘤胃菌科(HBW和LBW组分别为10.32%和9.98%)和未分类的毛螺菌科(HBW和LBW组分别为6.33%和4.53%)。PCoA分析显示,肠道微生物在杜洛克仔猪个体之间差异较小,但两组间在微生物组成上存在一些显著差异的菌属。在属水平上,HBW组猪中北里孢菌属、g_norank_o_Tremblayales、未命名的丹毒丝菌属、未分类的普雷沃氏菌科和粪芽孢菌属的丰度显著高于LBW组(P<0.05),棒状杆菌属在LBW组中的丰度显著高于HBW组(P<0.05)。此外,KEGG通路差异分析发现,倍半萜类化合物生物合成、胰岛素信号通路、抗坏血酸和醛酸代谢及安沙霉素类合成的途径显著富集于HBW组(P<0.05),分泌系统途径显著富集于LBW组(P<0.05)。本研究结果有助于理解猪早期肠道微生物与宿主间的相互作用和猪的健康养殖。     

基于16S rRNA高通量测序技术分析草原红牛瘤胃微生物多样性和功能预测的研究

为系统探讨草原红牛瘤胃内的微生物多样性及其功能,本试验利用16S rRNA基因高通量测序技术检测分析草原红牛（20月龄左右,均重为577.5 kg）瘤胃液样本菌群结构并进行PICRUSt功能预测。结果显示:通过Illumina Miseq测序平台共获得35 848条优质序列,聚类分析得到387个操作分类单元（OTU）,经分类学鉴定分属15个门、20个纲、25个目、41个科及110个属;厚壁菌门（Firmicutes）和拟杆菌门（Bacteroidetes）为优势菌群,所占比例分别为50.09%和41.11%;基于属的组成,依次为普雷沃菌属（Prevotella）15.20%、未知属f型拟杆菌目（norank_f_Bacteroidales）BS11菌群8.66%、瘤胃菌科（Ruminococcaceae）NK4A214菌群6.96%、理研菌科（Rikenellaceae）RC9菌群5.56%、未知属（Christensenellaceae）R-7菌群4.01%、瘤胃球菌属₂（Ruminococcus₂）3.33%等;16S rRNA基因组的PICRUSt功能预测结果显示,瘤胃内菌群功能主要集中在碳水化合物转运及代谢,表面草原红牛体内含有大量的纤维素和木质素降解酶基因。综上,基于16S rRNA基因的高通量测序技术全面揭示了草原红牛瘤胃菌群的多样性,且预测其含有丰富的蛋白质分解、木质纤维素降解酶系,为探索草原红牛瘤胃微生物的认知提供了基础,也为挖掘其他重要营养生理功能相关的瘤胃微生物功能基因提供了参考。     

基于16S rRNA测序分析高低饲料转化率猪粪便微生物的组成差异

提高猪饲料转化率是目前猪育种工作的重点,本研究对384头三元杂交母猪的FCR值进行排序,从中选取两端高饲料转化率(HFCR组)和低饲料转化率(LFCR组)猪各10头,采集粪便进行微生物16S rRNA基因测序分析。结果表明,LFCR组的Richness指数、Chao1指数、ACE指数均极显著高于HFCR组(P<0.01)。通过LEfSe分析,在属水平上筛选两组间具有显著差异的微生物,其中,在LFCR组筛选LDA>3的微生物主要是Prevotella＿1、Treponema＿2、Prevotellaceae＿NK3B31＿group、Eubacterium＿coprostanoligenes＿group、p-1088-a5＿gut＿group、Prevotella＿9、阿克曼菌属、Prevotellaceae＿UCG＿009、Ruminococcaceae＿UCG-014、Ruminococcaceae＿NK4A214＿group,在HFCR组LDA>3的微生物主要是巨球藻属、小类杆菌属、链型杆菌属、假丁酸弧菌属、柯林斯氏菌、Solobacterium、罕见小球菌属、链...     

基于16S rRNA测序分析PRRSV感染仔猪肺和肠道中微生物菌群的变化

本试验旨在探究猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)感染对仔猪肺、肠道中的菌群影响以及肺和肠道的组织学变化。试验选取35日龄断奶健康仔猪14头,适应性饲养7 d,随机分为感染组(n=7)和对照组(n=7),感染组仔猪接种2 mL 1×10⁵ TCID₅₀·mL^-1PRRSV-JTS毒株病毒液,对照组仔猪接种2 mL DMEM培养基。感染组3头仔猪分别于10、12和19 d死亡,将存活的感染组与对照组仔猪于21 d后处死,并取肺、不同肠段组织样品以及肠道内容物。采用免疫组化染色和HE染色观察肺和肠道组织病理学变化,并基于16S rRNA基因的高通量测序分析仔猪肺和各肠段菌群结构。结果表明,感染组仔猪肺部和肠道均有病毒分布;与对照组仔猪相比,感染组仔猪肺部和肠道均存在明显的炎症反应。微生物组成结构及多样性分析显示,感染组仔猪Chao1、ACE指数在肺中上升,在仔猪各肠段中下降,Shannon、Simpon指数在肺中升高,在...     

基于16S rDNA高通量测序方法检测猪舍环境微生物群落多样性

[目的]研究猪舍环境微生物群落多样性,了解不同类型猪舍环境中细菌群落的结构差异以及潜在的致病菌属。[方法]利用空气沉降法采集规模化猪场妊娠猪舍、保育猪舍、分娩猪舍的环境微生物样本,每种类型猪舍选取3栋猪舍采样,共9个样本。采用Illumina Miseq技术,以细菌16S rDNA的V3～V4变异区为对象进行高通量测序;采用QIIME2软件对测序数据进行分析,比较不同类型猪舍环境微生物群落组成。[结果]在97%相似度阈值下9个样本共得到OTU 29 126个,涵盖31门66纲160目320科899属1 831种的细菌。分娩猪舍以及保育猪舍的Chao1指数、Shannon指数及谱系多样性指数整体高于妊娠猪舍。变形菌门（Proteobacteria）、拟杆菌门（Bacteroidota）、厚壁菌门（Firmicutes）和放线菌门（Actinobacteriota）为猪舍环境的四大优势菌群;变形菌门在3个类型的猪舍中相对丰度均最高,厚壁菌门、放线菌门以及拟杆菌门相对丰度在不同类型猪舍中差异较大。不动杆菌属（Acinetobacter）、棒杆菌属（Corynebacterium）、寡养单胞菌...     

16S rRNA、23S rRNA及16S～23S rRNA基因在细菌分离与鉴定中的应用

在细菌的进化过程中,rRNA结构既具保守性又具高变性.保守性反映生物物种的亲缘关系,高变性则揭示生物物种的特征核酸序列,rRNA是属种鉴定的分子基础.因此,可以利用保守区设计通用引物扩增细菌的相应靶序列,再利用可变区的差异鉴别菌种,文章就16S rRNA、 23s rRNA和16S～23S rRNA基因在细菌分离与鉴定中的应用做以下综述.     

Analysis of Rumen Microbial Diversity and Functional Prediction of Chinese Simmental Cattle Based on 16S rRNA High-throughput Sequencing Technology

To systematically explore the microbial diversity and function in the rumen of Chinese Simmental cattle,this experiment used 16S rRNA gene high-throughput sequencing technology to detect and analyze the rumen fluid samples of Simmental cattle (about 20 months old,the average weight was 550 kg),and its function was predicted by PICRUSt.The results showed that a total of 66 712 high-quality sequences were obtained from the Illumina Miseq sequencing platform,and 1 797 operational classification units (OTU) were obtained by cluster analysis.The taxonomic identification was divided into 13 phylums,20 classes,22 orders,33 families and 59 genuses;Firmicutes and Bacteroidetes were dominant bacteria,accounting for 62.46% and 22.45%,respectively;Based on the genus composition,[Eubacterium]_coprostanoligenes_group (9.16%),Ruminococcus_2 (7.90%),norank_f__Bacteroidales_RF16_group(6.23%),norank_f__Ruminococcaceae(4.79%),Rikenellaceae_RC9_gut_group(4.72%),norank_f_Bacteroidales_BS11_gut_group(4.49%),Ruminococcus_1(4.43%),etc.The PICRUSt function of the 16S rRNA genome predicted that the rumen flora function was mainly concentrated in carbohydrate transport and metabolism,and might contain a large amount of cellulose and lignin degrading enzyme genes.In summary,the high-throughput sequencing technology based on 16S rRNA gene fully revealed the diversity of rumen bacteria in Simmental cattle,and predicted that it was rich in protein decomposition and lignocellulose degrading enzymes.The basis of microbial cognition provided a reference for further research on rumen microbial functional genes closely related to some important nutritional and physiological functions.     

基于16S rRNA高通量测序技术分析中国西门塔尔牛瘤胃微生物多样性和功能预测的研究

为系统探讨中国西门塔尔牛瘤胃内的微生物多样性及其功能,本试验利用16S rRNA基因高通量测序技术检测分析西门塔尔牛(20月龄,平均体重约为550 kg)瘤胃液样本菌群结构并进行PICRUSt功能预测。结果显示,两组样本通过Illumina Miseq测序平台共获得66 712条优质序列,聚类分析得到1 797个操作分类单元(OTU),经分类学鉴定分属13个门20个纲22个目33个科及59个属;厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)为优势菌群,所占比例分别为62.46%和22.45%;基于属的组成,依次为粪杆菌真核菌群([Eubacterium]_coprostanoligenes_group,9.16%)、瘤胃球菌属_2 (Ruminococcus_2,7.90%)、未知属f型拟杆菌目RF16菌群(norank_f__Bacteroidales_RF16_group,6.23%)、未知属f型瘤胃菌科(norank_f__Ruminococcaceae,4.79%)、理研菌科_RC9菌群(Rikenellaceae_RC9_gut_group,4.72%)、未知属f型拟杆菌目BS11菌群(norank_f_Bacteroidales_BS11_gut_group,4.49%)、瘤胃球菌属_1(Ruminococcus_1,4.43%)等;16S rRNA基因组的PICRUSt功能预测发现,瘤胃菌群功能主要集中在碳水化合物转运及代谢上,可能含有大量的纤维素和木质素降解酶基因。综上,基于16S rRNA基因的高通量测序技术全面揭示了西门塔尔牛瘤胃菌群的多样性,并预测其含有丰富的蛋白质分解、木质纤维素降解酶系,为探索西门塔尔牛瘤胃微生物奠定基础,也为进一步挖掘与某些重要营养生理功能密切相关的瘤胃微生物功能基因提供参考。     

应用16S rRNA基因测序法鉴定兔支气管败血波氏杆菌的研究

应用16S rRNA基因测序法对兔支气管败血波氏杆菌Bb-1株进行了16SrRNA基因序列分析。结果表明,能够扩增出与预期结果相符合的片断。经Blastn比较,分离菌与兔支气管败血波氏杆菌RB50株（BX640449）同源性为99．8％。进一步的生化实验鉴定表明,Bb-1株生化反应结果符合兔支气管败血波氏杆菌生化反应特征。综合各种实验结果表明,分离菌Bb-1株为兔支气管败血波氏杆菌。     

猪附红细胞体16S rRNA基因的序列测定和系统进化分析

从确诊为猪附红细胞体感染的猪场，无菌采集血样，抽提猪附红细胞体基因组DNA，采用真细菌的通用引物进行16S rRNA基因扩增，对扩增产物进行克隆和测序。从3个地理位置不同的猪场均成功地扩增出长度为1469bp的核苷酸序列。系统进化分析表明，3个猪场样品所测序列一致性达99．52％以上，具有相同的基因型，但与国外报道的猪附红细胞体Illinois株同源性为95％，属于同一基因群，但基因型不同；所有种类的附红细胞体和血巴尔通氏体组成同一进化分支，这类血营养菌与支原体科，支原体属的病原最靠近(75％)，而与立克次氏体目的病原较远(70％)。上述研究证实，广东所流行的猪附红细胞体是一种新基因型的猪附红细胞体，建议命名为猪附红细胞体广东株型；为反映进化关系，猪附红细胞体和其它血营养菌应划归于支原体科的支原体属。     

Analysis of the Cecum Microbial Diversity of Different Pig Breeds by Illumina Amplicon Sequencing of 16S rDNA Tag

To study the diversity and composition of the bacterial community from cecum samples from Yacha pig, Qingyu pig and Wujin pig, the investigation was designed to reveal mechanism of roughage resistance. Illumina amplicon sequencing of 16S rDNA Tag was used to analyze the cecum microbial diversity of Yacha pig, Qingyu pig and Wujin pig at 90 kg liveweight. The results indicated that the core flora of the three breeds were Bacteroidetes and Firmicutes at the phylum level and Prevotella and Bacteroides at the genus level. Meanwhile bacteria associated with cellulose decomposition were found in all three breeds, in which the number of Fibrobacter and Clostridium in Yacha pig were significantly higher than that in Qingyu pig and Wujin pig (P<0.05),and the number of Prevotella in Yacha pig was significantly higher than that in Wujin pig (P<0.05). The number of Bacteroides in Qingyu pig was significantly higher than that in Yacha pig (P<0.05).The number of Spirochaetes in Qingyu pig was significantly higher than that in Wujin pig (P<0.05). It could conclud that the composition of the cecum bacteria community was similar in above pig breeds, but there were significant differences in distribution and quantity. According to analysis of microbial related to cellulose digestibility, Yacha pig was the strongest among the three breeds, followed by the Qingyu pig and Wujin pig.     

获得性16S rRNA甲基化酶的研究进展

外源获得性16S rRNA甲基化酶基因广泛分布于革兰阴性细菌中,介导对多种氨基糖苷类药物的高水平耐药。该酶能将S-腺苷-L-甲硫氨酸的甲基添加到16S rRNA氨酰tRNA识别位点的特异性核苷酸上,从而干扰氨基糖苷类药物与靶位点的结合。16S rRNA甲基化酶基因通常由转座子等活动性遗传元件介导并嵌入到可转移的质粒或染色体中,从而导致耐药基因广泛而迅速地传播。更令人担忧的是,16S rRNA甲基化酶基因经常与bla_NDM-1、bla_CTX-M和qnrB1等其他耐药基因偶联,介导对β-内酰胺类药物和氟喹诺酮类药物的多重耐药。对于多重耐药16S rRNA甲基化酶阳性菌引发的感染,治疗方法非常有限。迄今为止,至少已有30个国家和地区对16S rRNA甲基化酶进行了相关报道,由此可见,16S rRNA甲基化酶基因的全球传播正成为一个全球重大公共卫生问题。     

温氏附红细胞体部分16S rRNA基因的序列测定和分析

从确诊为附红细胞体感染的黄牛无菌采集血样，抽提附红细胞体基因组DNA，用实验设计的能扩增多种动物血营养菌部分16SrRNA基因的通用引物进行PCR扩增，结果扩增出大小约为370bp的DNA片段。PCR产物序列测定和系统进化分析显示，实验获得的核苷酸序列为温氏附红细胞体的16SrRNA基因，与国外报道的温氏附红细胞体的同源性为97％。反映出不同地理株的温氏附红细胞体存在一定的遗传差异，为牛附红细胞体病的诊断和分子流行病学研究提供科学依据。     

桑黄化型萎缩病病原体16S rRNA基因的序列分析

用PCR法克隆了中国桑黄化型萎缩病病原植原体的 16SrRNA基因 ,并进行了序列分析。结果表明 :克隆的基因大小为 1372bp ,与日本桑萎缩病病原植原体 16SrRNA的同源性高达 99 85 % ,只存在个别碱基的突变。Blastj检索结果显示与中国桑黄化型萎缩病病原体 16SrRNA的基因同源性高达 99%的有来源于玉米、洋葱、土豆等5 0多种其它植物的植原体 16SrRNA基因 ,表明植物萎缩病病原体 16SrRNA基因具高度保守性。     

Effect of Fructooligosaccharide on the Structure and Diversity of Rumen Bacterial Community in Dairy Cow by 16S rDNA Sequencing Technology

This experiment was aimed to study the effect of fructooligosaccharides on the structure and diversity of rumen bacterial community in dairy cows by 16S rDNA sequencing technology.Four lactating dairy cows with similar lactation stages and the same parity were randomly divided into two groups using two-phase cross design method.The cows in control group were fed with basal diet and that in test group were fed with the basal diet with 60 g/(d·head) fructooligosaccharides.The experiment was designed through 2×2 cross-over test with 21 d in each stage (14 d for pre-experiment and 7 d for test) and 14 d transitional period of cross test.The rumen fluid samples were collected at 0 (before feeding),2,4,6,9 and 12 h after feeding by cattle catheter.Each phase was collected continuously for 3 days,and the structure and diversity of rumen bacterial community were measured by 16S rDNA sequencing technology.The results of Alpha diversity indexes (Simpson,Shannon),richness indexes (Chao,Ace) and Beta diversity profile showed that the addition of fruitooligosaccharide decreased the bacterial diversity in the rumen of dairy cows.And the results of 16S rDNA sequencing showed that according to the analysis at phylum level,Bacteroidetes,Firmicutes and Proteobacteria accounted for more than 95% of the total bacterial population in cow rumen of two groups.The addition of fructooligosaccharides significantly decreased the abundance of Bacteria_NA (P < 0.05),and the abundance of Synergistetes showed an increasing trend (P=0.075).According to the analysis at genus level,fructooligosaccharides significantly increased the abundance of the Pseudomonas (P < 0.05),extremely significantly increased the abundance of Bacteroides (P < 0.01),while the abundance of Dehalobacterium significantly decreased (P < 0.05).Compared with the control group,the abundance of Ruminococcus,Butyrivibrio,Succiniclasticum and Lachnospiraceae_NA in test group was increased by 80.0%,7.5%,127.9% and 20.0%,respectively,but these differences were not significant (P > 0.05).Under the test conditions,the addition of fructooligosaccharides had a certain effect on the diversity and structure of rumen bacterial flora in dairy cows,which significantly increased the abundance of Pseudomonas which unfermented sugar,extremely significantly increased the abundance of Bacteroides which used carbohydrates as a source of fermentation,had an accelerating effect on the abundance of rumen fiber degrading bacteria.