16S rRNA Gene-Based Metagenomic Analysis of Soil Bacterial Diversity in Brazzaville, Republic of the Congo

Soil contains a great diversity of microorganisms, among which are bacteria. This study aimed to explore bacterial diversity in soil samples in Brazzaville in the Republic of the Congo. Environmental DNA was extracted. The illumina MiSeq sequencing was held and the diversity indices have been computed. Illumina MiSeq sequencing revealed 21 Phyla, four of which were abundant: Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes. Soil microbial communities in the studied samples were phylogenetically diverse but with a stable community structure. 17 classes are represented with relative abundances of Rihzobiales, Bacillales, Actinomycetales and Acidobacteriales. 40 families, the Alphaproteobacteria, the Bacilli and the 12 Actinobacteria. 83 orders among which the Rhizobiales are the most abundant followed by Bacillales and the least abundant followed by the Flavobacteriaceae. Of the 28 genera listed, the Bradyrhizobium is the most dominant in Mw3 and Mw4. 25 listed species, Bradyrhizobium, Bacillus, Actinoplanes, and Candidatu coribacter Acidobacterium are the most abundant species. The Shannon indices of Mw3 and Mw4 are equal, the H’max of Mw4 is greater than the H’max of Mw3. The Simpson index of Mw4 is equal to the Simpson index of Mw3, and the Pielou index (J) of Mw4 is less than the R of Mw3, but very close. This study opens interesting perspectives on the knowledge and exploitation of telluric bacteria in several areas of life.

High-throughput sequencing of 16S r RNA amplicons characterizes gut microbiota shift of juvenile sea cucumber Apostichopus japonicus feeding with three antibiotics

Sea cucumber Apostichopus japonicus is an important marine economic species in Asian countries due to its profound nutritional and medicinal value. So far, with the rapid development of intensifi ed artifi cial aquaculture of sea cucumbers, the use of antibiotics is still an inexpensive and dispensable way to treat pathogenic infections, especially during the nursery phase. However, there is little information on the eff ects of antibiotics on the intestinal microbiota of sea cucumber. Therefore an Illumina based sequencing method was used to examine the intestinal bacterial composition of juvenile A . japonicas following diets with three typical antibiotics (tetracycline, erythromycin, and norfl oxacin) under 15, 30, and 45 d. The fi ndings reveal that diff erent antibiotics have distinct eff ects on the growth performance of juvenile sea cucumbers. However, the richness and diversity of microbiota were barely aff ected by antibiotics but the community composition alterations indicated that the three antibiotics exhibited their respective patterns of reshaping the intestinal bacteria of juvenile sea cucumbers. In common, the abundance of some sensitive genera with helpful functions, such as Thalassotalea , Shewanella , Sulfi tobacter , and Halomonas decreased signifi cantly with exposure to antibiotics and the abundance of multiple potential pathogenic- and suspected antibiotic-resistant microorganisms like Arcobacter , Leucothrix , and Clostridium_sensu_stricto_1 was found increased signifi cantly in the antibiotic groups. These results suggest that low doses of antibiotics could aff ect the composition of the intestinal microbiota of sea cucumbers and might increase the risk of infection of the hosts. This study could help us to explore how antibacterial compounds modify the gut microbiota of sea cucumbers and provide theoretical guidance in hatchery management by scientifi c antibiotic use in sea cucumber mariculture.

基于16S rRNA测序分析阻塞性睡眠呼吸暂停患者肠道靶标菌群的变化

目的分析阻塞性睡眠呼吸暂停(OSA)患者和健康人群肠道菌群的差异,探讨肠道菌群在OSA发病中的作用及意义。方法随机纳入2022年1月~12月就诊于本院诊断为OSA的患者39例作为OSA组,健康志愿者20例作为对照组。收集两组人群的粪便标本,通过16SrRNA高通量测序分析其微生物组成,分析两组人群肠道菌群之间的Alpha多样性、Beta多样性、物种差异与标志物种和差异生物功能代谢通路功能预测分析。结果Alpha多样性分析显示,OSA组的物种多样性指数Shannon和Simpson、物种丰度指数Observedspecies及菌群均匀度指数Pielou均低于对照组(P<0.05);Beta多样性分析显示,两组间群落结构存在差异(P<0.05)。OSA组肠道菌群群落结构上与对照组存在差异,潜在致病菌属如假单胞菌属、巨单胞菌属等菌群丰度增加(P<0.05)。LEfSe分析结果显示,与对照组相比,OSA组假单胞菌属、巨单胞菌属、梭杆菌属丰度升高(P<0.05)。关联网络分析结果显示,影响宿主稳态的为差异标志菌群;随机森林分析和ROC曲线结果显示,假单胞菌属是具有重要鉴别意义的生物标志菌属。差异代谢通路预测功能显示,维持肠道菌群稳态起主要作用功能的是生物合成功能,假单胞菌属参与了芳香族生物胺降解和酮葡萄糖酸代谢。结论OSA患者存在肠道菌群紊乱,肠菌中假单胞菌属可能通过参与物质代谢影响OSA发生发展,可望作为防治OSA的潜在肠菌靶标。

Cloning and Sequence Analysis of 16S rRNA and COI Gene in Mitochondrial DNA of Scortum barcoo

[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.

基于16S rRNA基因测序法分析勃起功能障碍患者口腔微生物多样性

目的通过16S rRNA高通量测序分析勃起功能障碍(erectile dysfunction,ED)患者和正常健康人的口腔菌群结构差异,探讨ED与口腔菌群的关系。方法选择贵州中医药大学第一附属医院泌尿外科门诊就诊的年龄段在20~65岁的ED患者(ED组)与健康者(正常对照组),各15例,收集唾液样本分别进行16S rRNA高通量测序,对各组唾液样本菌群的结构进行比较和分析。采用独立样本t检验、非参数检验、χ^(2)检验进行统计分析。结果ED组和正常对照组唾液中菌群的核心优势菌种组成相似,在菌属水平上优势菌属前10位分别为梭杆菌属、雷尔氏菌属、奈瑟氏菌属、嗜血杆菌属、普雷沃-7菌属、韦荣氏球菌属、异戊酸杆菌、链球菌属、卟啉菌属、放线菌属。两组唾液样本中不同菌群的丰度差异均有统计学意义(均P<0.05)。ED组中的链球菌属的丰度高于正常对照组[8.59(4.13,11.75)%比4.23(3.25,7.48)%],梭杆菌属的丰度低于正常对照组[9.38(5.61,14.91)%比14.13(7.92,19.03)%],两组比较差异均有统计学意义(均P<0.05)。结论ED患者与健康人的口腔菌群存在着相同的核心组成骨架,而菌群结构却存在一定的差异,这或许和ED的发生发展有着密切联系,但其中的因果关系及其相关机制还有待进一步深入探讨。

Genetic Diversity in Populations of Sepiella maindroni Using 16S rRNA Gene Sequence Analysis

Part of the 16S rRNA gene is amplified with PCR and sequenced for 5 populations of com-mon Chinese cuttlefish Sepiella maindroni: three from the South China Sea, one from East China Sea and one from Japan. The result shows that a total of 5 nucleotide positions are found to have gaps or insertions of base pairs among these individuals, and 13 positions are examined to be variable in all the sequences, which range from 494 to 509 base pairs. All of the individuals are grouped into 7 haplotypes (h1-h7). No marked genetic difference is observed among those populations. All of the individuals from Nagasaki belong to hl and the h3 haplotype is found only in the coastal waters of China. A(?)G transition in Nucleotide 255 is suggested to be taken as a kind of genetic marker to identify the populations distributed in East-South China Sea and the Nagasaki waters of Japan.

Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on ge- netic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the rela- tionship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K. bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

16S rRNA Gene Sequence Analysis of Snow Leopard, Gray Wolf, Horse and Bactrian Camel in Mongolia

In this study, mitochondrial 16S rRNA sequences of snow leopard, gray wolf, domestic horse and Bactrian camel inhabited or domesticated in Mongolian territory were obtained by using polymerase chain reaction （PCR） based on universal primers for 16S rRNA （F-5＇-AACGAGCCTGGTGATA-3＇ and R-5＇-CTCCGGTCTGAACTCAGATCACGTA-3＇）. The 16S rRNA sequence was 1,048 bp to 1,086 bp in length, and each sequence was compared to other related species （Felidae, Camelidae, Equidae and Canidae） by using NCBI Basic Local Alignment Search Tool （BLAST）. Results showed that sequences were highly similar to sequences in GenBank database （93%-99%）. Then phylogenetic analysis was performed based on about 1,100 bp sequence of 16S rRNA for Panthera uncia, Canis lupus, Equus caballus, Camelus bactrianus and other related species. The result revealed that P. uncia and P. leo were sister species, C. bactrianus and C. ferus were more closely related species, and wolf and dog were the almost similar species. This finding could be important for designing species specific primers for PCR based analysis of animal species identification and forensic veterinary medicine.

16S rRNA测序技术在肠道微生物中的应用研究进展

16S rRNA测序是高通量测序依赖的肠道微生物研究方法之一,该方法可以对肠道微生物中的所有菌种进行精确定量,因此正逐渐成为研究肠道微生物菌种丰度变化的主流。肠道微生物16S rRNA测序的应用过程中有两个问题至关重要,一是如何根据需要选择测序方案;二是面对高通量测序得到的海量数据,如何进行生物信息学分析,以得到具有生物学意义的结果。从测序平台、测序片段、测序数据量的选择3个方面讨论了如何选择测序方案,并从序列聚类与注释、群落结构分析、关键分类单位的筛选与功能分析等方面对目前常用的生物信息学分析手段进行综述。

基于16s rRNAs高通量测序分析糖尿病足骨髓炎感染骨组织中的病原微生物

目的利用16s rRNA高通量测序技术分析糖尿病足骨髓炎(DFO)感染骨组织中病原微生物特点,为临床感染病原菌的鉴定及治疗提供快速、准确的方法。方法收取2016年9月~2017年4月于本科室住院的16例DFO患者清创术中获取的感染骨标本,分别利用16s rRNA高通量测序技术和血培养分析仪进行病原微生物的鉴定,分析16s rRNA测序结果中DFO的菌群特点,并与培养结果相比较。结果 16s rRNA测序显示DFO骨组织病原微生物多样性较大,分布较为均匀,共获得优势菌属20种,占所有菌属的87.00%,其中Prevotella是丰度最大的菌属。两种鉴定方法结果均显示DFO病原菌以革兰氏阴性菌为主。与培养法相比,16s rRNA测序阳性率较高(100%vs 88.24%),平均每个样本病原菌数较多(12.56 vs 1.50),革兰氏阴性菌所占的比例较高(67.16% vs 50.00%)。此外,16s rRNA测序结果覆盖了除Escherichia coli、Serratia marcescens及Enterobacter cloaca外的所有培养病原菌结果,甚至有高达13种菌属不存在于培养结果,其中Anaerococcus、Veillonella、Bacteroides、Fusobacterium、Porphyromonas、Finegoldia、Prevotella、Peptostreptococcus、Parvimonas、Peptoniphilus和Bulleidia均为专性厌氧菌或严格厌氧菌,而培养结果中并无厌氧菌的出现。但培养结果显示,DFO中多重耐药菌的比例高达58.33%。结论 16s rRNA高通量测序能较好展示DFO骨组织中菌群微生态的多样性及丰度特点。DFO骨组织病原微生物多样性大,分布均匀,但优势菌属分布离散,以革兰氏阴性菌为主。与培养法相比,16s rRNA测序对病原菌的鉴定简单而准确,尤其在对革兰氏阴性菌和厌氧菌鉴定方面有显著的优势,可快速而准确地指导DFO感染治疗。

中国对虾16SrRNA基因序列多态性的研究

利用PCR技术扩增获得中国对虾 (Penaeuschinensis)线粒体DNA的 16SrRNA基因片段 ,通过测定该基因片段的序列 ,分析了取自我国烟台、长岛、青岛近海和宁波养殖的 17只中国对虾遗传多态性。结果发现 ,不同地理种群存在丰富的DNA序列多态性 ,17只个体具有 17种基因型 ,在扩增的长为 5 2 3bp的基因片段中 ,共检测到 3 7个多态性核苷酸位点 ( 7 0 7% )。UPGMA法构建的分子系统树表明不同地理种群中国对虾存在一定程度的遗传分化 ,长岛群体与烟台群体遗传关系较近 ,宁波群体次之 ,青岛群体为相对独立的一支。

基于16S rRNA高通量测序的西藏农、牧区牦牛酸奶菌群多样性分析

运用高通量测序技术,对西藏农、牧区牦牛酸奶中细菌多样性及其差异进行分析,并探究环境因素对其差异形成的影响。样本层级聚类及主坐标分析结果显示农、牧区的样本菌群结构具有明显差异;多级物种差异判别分析及组间差异分析表明,农、牧区牦牛酸奶菌群的优势属均为厚壁菌门(Firmicutes)下的乳杆菌属(Lactobacillus)、乳球菌属(Lactococcus)及链球菌属(Streptococcus),上述3个属及其余7个丰度相对较低的属在不同区域牦牛酸奶中具有显著差异(P<0.05);对环境因子的回归分析和基于距离的冗余分析显示海拔高度对样本间差异的解释度大于年平均气温。本研究为进一步揭示自然环境与青藏高原发酵食品中微生物多样性之间的关系、筛选优良菌种提供参考。

福建华溪蟹线粒体DNA COI和16S rRNA基因序列的遗传多样性

目的探讨福建华溪蟹(Sinopotamon fukienense)的遗传多样性。方法采用PCR结合DNA测序技术,测定S.fukienense的线粒体COI和16SrRNA基因序列的组成。经比对获得639bp长度的COI基因序列和526bp的16SrRNA基因序列,以对比分析S.fukienense的遗传多样性。结果 S.fukienense基于COI基因核苷酸多样性(Pi)为0.048 4,高于其基于16SrRNA基因核苷酸多样性(Pi)为0.021 6。同时,S.fukienense基于COI基因单倍型间的平均遗传距离(P)为0.048,大于其基于16SrRNA基因单倍型间的平均遗传距离(P)0.026。结论 COI序列在分析S.fukienense遗传异变时的作用更优于16SrRNA基因序列。

基于16S rRNA技术分析石珍安神汤对脱髓鞘小鼠肠道菌群多样性的影响

目的基于16S rRNA技术研究石珍安神汤(Shi-Zhen-An-Shen-Tang,SZAST)对脱髓鞘模型小鼠肠道菌群多样性的影响,并探讨该复方治疗精神分裂症的作用机制。方法SPF级5周龄雄性C57BL/6小鼠30只,采用数字表法随机分为普通饲料对照组、模型组、中药低剂量组、中药中剂量组、中药高剂量组、喹硫平组,每组5只。适应性喂养2周后,对照组正常饮食,其他组食用混合了0.2%(质量分数)双环乙酮草酰二腙(cuprizone,CPZ)的特殊饲料。食用特殊饲料4周后,SZAST组给予SZAST灌胃2周(低浓度组5.5 g·kg^-1·d^-1,中浓度组11.0 g·kg^-1·d^-1,高浓度组16.5 g·kg^-1·d^-1),模型组以0.9%(质量分数)氯化钠注射液灌胃2周(10 mL·kg^-1·d^-1),喹硫平组按10 mg·kg^-1·d^-1给予喹硫平溶液灌胃。2周后小鼠麻醉取盲肠内容物。以Illumina MiSeq为测序平台,对其16S rRNA V3-V4区的肠道菌群可操作分类单元(operational taxonomic units,OTUs)数量,alpha与beta多样性,丰富度指数与多样性指数以及差异菌与菌属进行综合性分析与评价。结果石珍安神汤可改善脱髓鞘小鼠OTUs数量及alpha与beta多样性的失调,对脱髓鞘小鼠的β-变形菌纲、伯克菌目、拟杆菌科、产碱杆菌科、毛螺菌属-NK4A136等差异菌分布结构均产生回调作用。结论石珍安神汤对脱髓鞘模型小鼠的异常菌群多样性起到调节作用,并通过16S rRNA技术揭示了肠道菌群在白质损害导致精神分裂症中的相关病理机制。

基于16S rRNA高通量测序技术的肠道菌群多样性推断死亡时间

目的采用16S rRNA高通量测序技术探究大鼠肠道菌群变化与死亡时间(postmortem interval,PMI)之间的关系。方法大鼠腹腔麻醉致死后置于16℃,提取死后0、1、2、3、5、7、9、12、15、18、21、24、27和30 d共14个时间点的盲肠内容物DNA,采用16S rRNA高通量测序技术,检测大鼠盲肠内容物中的肠道菌群,对数据进行多样性及差异性分析。结果死后30 d内大鼠肠道微生物菌群总数未发生明显变化,但菌群多样性呈上升趋势。在死后13个时间点共筛选出119个具有显著差异的细菌群落。构建全部时间点、9 d前、12 d后PMI推断的偏最小二乘(partial least squares,PLS)回归模型,其决定系数(R2)分别为0.795、0.767和0.445;交叉验证均方根误差分别为6.57、1.96和5.37 d。结论利用16S rRNA高通量测序技术探究死后30 d内肠道菌群的变化规律,其组成和结构出现了明显的变化,且建立的PLS回归模型表明PMI与肠道菌群之间高度相关,呈一定时序性变化。

石化污泥优势菌群的高通量分析及16S rRNA基因鉴定

针对石化污泥中的细菌进行了454高通量分析,共得到11 488条序列.发现在相似度97%的水平上,样品的OTU丰度最高;经测序分析,石化污泥中共有细菌235个属,其中短小盒菌属最多,占到整个细菌菌群的28.39%,芽孢杆菌属占14.43%,属种丰度1.5%～10.0%的有9个属(占28.99%).这11个属被确定为优势菌群.通过对污泥优势菌群的分离培养,依据16S r RNA基因鉴定该11属有22个种,并用16S r RNA基因序列比对建立了优势菌株系统发育树.该优势菌群能分解石化污水污泥中的工业污染物,并以此污染物为营养和能源,进行相对旺盛的生命活动,成为对石化污染水体进行生物修复的宝贵菌种资源.

16S rRNA高通量测序研究柴芪汤对代谢综合征大鼠肠道菌群的影响

目的:采用16S rRNA高通量测序技术分析柴芪汤对代谢综合征(MS)大鼠肠道菌群的影响。方法:通过Illumina Miseq PE300型高通量测序仪对大鼠粪便肠道菌群进行16S rRNA基因V3-V4可变区检测,运用Usearch、Mothur、LEfSe分析肠道菌群的组成结构及不同菌属相对丰度变化。结果:柴芪汤能够逆转MS大鼠肠道菌群物种多样性的下降,并纠正MS导致的菌群组成结构改变。研究发现MS大鼠的肠道菌群在菌属水平有57个菌属相对丰度发生显著变化,而柴芪汤能将其中的54个菌属恢复至正常水平。结论:从肠道微生态角度阐释了MS的可能病机,柴芪汤可以通过调节肠道菌群防治MS。

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30min~冷藏5h组中的优势菌属为金黄杆菌属(39.28%)、巨型球菌属(16.47%)、乳球菌属(9.61%),而在冷藏12~24h组中则以不动杆菌属(34.95%)、芽孢杆菌属(11.2%)、库特氏菌属(9.01%)为优势菌属。葡萄球菌属在刚挤出组中未检出,但随着冷藏时间的延长,其丰度呈上升趋势。可以得出,生鲜水牛乳从刚挤出至冷藏24h内,其微生物群落结构和组成呈动态变化,且此次测序中条件致病菌检出率较高,冷藏12h后样本组中细菌多样性显著高于其他组,由此可知生鲜水牛乳从挤出至加工前,低温贮藏时间越短越好,最好控制在5h内。

南疆维吾尔族儿童龋病及16S rRNA高通量测序菌斑微生物群落结构

目的:分析南疆维吾尔民族儿童患龋情况及微生物学角度研究龋病可能高发的原因。方法:通过分层整群抽样随机抽取南疆第三师51团5个连队幼儿园3～5岁维吾尔族儿童296名调查患龋情况及充填率,应用Illumina Miseq高通量测序技术分析高龋儿童、无龋儿童口腔微生物群落结构及多样性。结果:南疆第三师51团维吾尔族3～5岁儿童总患龋率83.4%、龋均(3.07±3.22)、龋补充填率2.07%。5岁组儿童患龋率、龋均明显高于3岁组,不同性别儿童患龋率和龋均之间差异均无统计学意义。无龋组与高龋组口腔菌斑呈微生物多样性,共98个outs,归属于12个门、18个纲、25个目、34个科、60个属。无龋组微生物多样性较高龋组丰富,Streptococcaceae,Porphyromonadaceae、Fusobacteria、Veillonellaceae、peptostreptococcaceae在两组所有样本中均存在,在高龋组中构成比高于无龋组。结论:南疆维吾尔族儿童患龋率明显高于国内平均水平,应加强对南疆地区群众的口腔资源投入,当地维吾尔族儿童中高龋和无龋人群口腔微生物群落结构存在一定的差异,高龋组中优势菌(Streptococcaceae,Porphyromonadaceae、Fusobacteria、Veillonellaceae、peptostreptococcaceae)对龋病发生发展的作用还有待进一步的研究。

基于16S rRNA高通量测序技术的生态学实验教学

利用微生物16S基因特定区域的通用引物对16S rRNA进行扩增,采用高通量二代测序技术,结合QIIME数据处理软件进行序列的筛选、比对和微生物鉴定,从而获得土壤中大量微生物物种OTU(Operational Taxonomic Unit)的分布信息。结果表明,该实验激发了学生的实验操作兴趣,拓展了本科生态学实验教学内容,有助于学生了解高通量测序技术在生态学研究领域的应用,提高学生综合实验能力。