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.

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.

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.

Correlation analysis of breast fibroadenoma and the intestinal flora based on 16S rRNA sequencing

Objective To analyze the characteristics of the intestinal microflora in patients with breast fibroadenoma using 16S ribosomal RNA(rRNA)high-throughput sequencing.Methods Fecal samples from 20 patients with breast fibroadenoma and 36 healthy subjects were randomly collected and analyzed using high-throughput sequencing technology for 16S rRNA V4 region sequencing,and the alpha diversity(Chao index,Shannon index)was calculated using Mothur(v.1.39.5)software.Beta diversity was analyzed using QIIME(v1.80).SPSS software(version 23.0)and the t-test of two independent samples were used to analyze differences in the abundance of bacteria between the two groups.Results Compared with that in the healthy control group,theαdiversity of the intestinal microflora in breast fibroadenoma patients increased,but the difference was not statistically significant(P>0.05).At the phylum level,significant differences were observed between the two groups.The abundance of Firmicutes was higher in the breast fibroadenoma group(P<0.05),whereas the abundance of Synergistetes was higher in the healthy control group(P<0.005).A total of five bacterial genera showed significant differences between the two groups:the breast fibroadenoma group showed higher levels of Bautia(P<0.005),Coprococcus(P<0.005),Roseburia(P<0.05),and Ruminococcus(P<0.005),whereas Sutterella was more abundant in the healthy control group than in the breast fibroadenoma group(P<0.05).Conclusion The diversity and abundance of the intestinal flora in patients with breast fibroadenoma are significantly different from those in healthy subjects,suggesting that an imbalance in the intestinal flora is correlated with the occurrence of breast fibroadenoma.

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.

12种石鲈科鱼类线粒体16S rRNA基因的部分序列分析

分析了5属12种石鲈科鱼类的线粒体16S rRNA基因部分序列特征,并参照RNA二级结构模型区分配对区和非配对区。结果表明,配对区对G有偏好(33.5%),非配对区对A有偏好(38.5%);与配对区相比,非配对区存在更多的变异和系统发育信息。从序列的Jukes-Cantor遗传距离结果看,石鲈科胡椒鲷属Plectorhynchus、矶鲈属Parapristipoma、石鲈属Hapalogenys、髭鲷属Pomadasys、Haemulon属5个属属间的差异水平都接近或超过了其与外类群科间的差异水平。从构建的ME系统发育树结果看,石鲈科鱼类分成二大分支,未能形成单一类群。在亲缘关系上,胡椒鲷属和矶鲈属较近,石鲈属和Haemulon属较近,髭鲷属与其它4个属较远,髭鲷属在鲈亚目中的分类地位可能应提高到科的水平。初步确定研究中选用的16S rRNA基因片段基本适用于石鲈科属间和属内种间关系的研究。

Identification of sika deer and red deer using partial cytochrome b and 12s ribosomal RNA genes

A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes sequences. 402 bp Cytb genes were achieved by PCR-sequencing using DNA extracted from 8 case samples, and contrasted with 27 sequences of Cytb gene downloaded from GenBank database. The values of three nucleotide distance between three suspected samples and sika deer were identical （0.026±0.006）, which was smaller than the smallest nucleotide distance between eastern red deer and sika deer （0.036）. Furthermore, phylogenetic analysis of sika deer and red deer indicated that the evidences located within the same cluster as sika deer. The evidences were sika deer materials. As the same way, other three suspected samples were derived from red deer. The results were further confirmed by phylogenetic and nucleotide distance analysis of 387 bp 12s rRNA gene. The method was powerful and less time-consuming and helpful to reduce the related cases with wildlife.

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。

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。

鲳亚目鱼类线粒体16S rRNA基因序列变异及其分子系统进化关系

为探讨鲳亚目鱼类的系统进化关系,通过测定中国沿海8种鲳亚目鱼类的线粒体16SrRNA基因部分序列,并结合GenBank上其他鲳亚目鱼类的同源序列,对其序列变异和分子系统进化树进行分析。结果表明,鲳亚目5科13属32种鱼类的16S rRNA基因序列的碱基组成为T 22.2%、C 24.5%、A 30.0%、G 23.3%;科间遗传距离为0.060～0.120,属间遗传距离为0.009～0.125,种间遗传距离为0.000～0.163;长鲳科位于系统进化树的基部,鲳科的鲳属处于系统进化树的顶端,无齿鲳科、方尾鲳科、双鳍鲳科与鲳科的低鳍鲳属和真鲳属聚类。结合形态学研究结果,认为:长鲳科是鲳亚目中最先分化的原始单系群;无齿鲳科和方尾鲳科为单系群,它们与非单系群的双鳍鲳科有较近的亲缘关系;鲳科为并系群,内部存在与地理区系相对应的2个分支,提示了该科鱼类早期的分化模式。同时,也对16S rRNA基因在鲳亚目鱼类系统进化研究中的适用性进行了剖析。

米尔顿姬小蜂线粒体16S rRNA与COⅠ基因片段序列测定及分析

【背景】米尔顿姬小蜂是一种入侵我国台湾地区的植食性小蜂,能够严重影响水果的产量和食用价值。目前在我国大陆没有分布,由于其个体微小,与近似种区别较小,通过传统的形态学分类方法难以鉴定,因此有必要研究其基因片段序列,探讨分子鉴定方法。【方法】利用PCR方法扩增并测定了米尔顿姬小蜂线粒体16SrRNA和COⅠ基因的部分序列,并对各序列的碱基组成进行了分析。然后根据COⅠ基因部分序列,利用DNAMAN的Maximum Likelihood方法构建了米尔顿姬小蜂与膜翅目其他科的系统发育树。【结果】16SrRNA基因的PCR扩增产物为426bp,COⅠ基因的PCR扩增产物为488bp。通过测序获得米尔顿姬小蜂16SrRNA和COⅠ基因部分序列,序列分析表明,16SrRNA和COⅠ基因的A+T含量均较高,存在较强的A+T偏向性。系统发育树显示,米尔顿姬小蜂与蚜小蜂科的Encarsia berlesei亲缘关系最近,与姬小蜂科的Chrysocharis nautius、C.eurynota亲缘关系较远。【结论与意义】本研究为米尔顿姬小蜂的分子鉴定提供了依据。

基于16S rRNA检测分析宫颈癌患者放射治疗前后肠道菌群变化特征

目的:探究电离辐射对宫颈癌患者肠道菌群的影响特点及菌群变化与放射性肠炎发生的相关性。方法:选取福建医科大学附属泉州第一医院2019年1月至2020年1月收治的30例宫颈癌患者,在其调强放射治疗的不同时间点收集其粪便样本,分析肠道微生态的生物标志物,用MoBio PowerSoil试剂盒提取肠道菌群DNA,用MiniSeq对肠道菌群16S rRNA的V4区进行测序及生物信息学分析。差异度采用Wilcoxon秩和检验等分析。结果:宫颈癌患者放射治疗后肠道微生物群的α多样性比放疗前高,同时放疗前后微生物群落组成(即β多样性)存在一定差异,但差异均无统计学意义(P>0.05);但是在宫颈癌患者放疗后微生物组成中普雷沃氏菌和链球菌较放疗前有显著变化(P<0.05)。结论:宫颈癌患者放疗前后微生物成分存在差异,但有待大规模的前瞻性队列研究证实。

16S rRNA序列分析技术对临床标本中疑难细菌的鉴定

目的应用16S rRNA序列分析技术鉴定临床少见或疑难细菌,提高细菌鉴定的准确性。方法收集临床少见或疑难细菌44株,提取DNA,采用通用引物进行PCR扩增及目的片段基因测序,并将测序结果在核酸数据库中比对分析以确定菌种。结果 44株临床少见或疑难细菌均扩增到16S rRNA目的基因片段并成功测序,40株(90.9%)鉴定到"种",4株(9.1%)鉴定到"属";其中常规方法与测序方法在"属"水平一致的有34株(77.3%),不一致的有10株(22.7%)。结论 16S rRNA序列分析技术可以准确、快速地鉴定临床少见或疑难细菌,可以作为临床细菌鉴定的重要方法之一。

基于16S rRNA序列的鳃金龟亚科5族种类的系统发育关系

测定了鳃金龟亚科部分种类的线粒体16SrRNA部分序列,运用MEGA 4.0、PAUP*4.0b10和MrBayes等软件,对5个族29个代表种的序列变异和系统关系进行了研究.序列变异分析结果显示:绢金龟族Sericini、哦鳃金龟族Hopliini、鳃金龟族Melolonthini的族内遗传距离分别为7.7%,11.3%,10.6%,族间遗传距离在12.6%～19.2%.最大似然树(ML)和贝叶斯树(BI)结果表明,所有种类分别聚集在所属的族下,哦鳃金龟族、绢金龟族分别为单系.齿爪鳃金龟属Holotrichia、云鳃金龟属Polyphylla、胸突鳃金龟属Hoplosternus、Hilyotrogus、双绺鳃金龟属Amphimallon、Hoplochelus、婆鳃金龟属Brahmina和皱鳃金龟属Trematodes聚在鳃金龟族分支下,没有形成明显的根鳃金龟族Rhizotrogini分支.齿爪鳃金龟属的非单系性与前人研究结果一致.皱鳃金龟属Trematodes和婆鳃金龟属Brahmina的代表种为姐妹种.绢金龟族的绢金龟属Serica和玛绢金龟属Maladera多数种类聚在本属分支下.证明16SrRNA序列可探讨鳃金龟亚科高级阶元的系统发育关系.

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.

Causal Relationship Between Gut Microbiota and Liver Cirrhosis:16S rRNA Sequencing and Mendelian Randomization Analyses

Background and Aims:Accumulating evidence highlights the association between the gut microbiota and liver cirrhosis.However,the role of the gut microbiota in liver cirrhosis remains unclear.Methods:We first assessed the differences in the composition of the bacterial community between CCl4-induced liver cirrhosis and control mice using 16S rRNA sequencing.We then performed a two-sample Mendelian randomization(MR)analysis to reveal the underlying causal relationship between the gut microbiota and liver cirrhosis.Causal relationships were analyzed using primary inverse variance weighting(IVW)and other supplemental MR methods.Furthermore,fecal samples from liver cirrhosis patients and healthy controls were collected to validate the results of the MR analysis.Results:Analysis of 16S rRNA sequencing indicated significant differences in gut microbiota composition between the cirrhosis and control groups.IVW analyses suggested that Alphaproteobacteria,Bacillales,NB1n,Rhodospirillales,Dorea,Lachnospiraceae,and Rhodospirillaceae were positively correlated with the risk of liver cirrhosis,whereas Butyricicoccus,Hungatella,Marvinbryantia,and Lactobacillaceae displayed the opposite effects.However,the weighted median and MR-PRESSO estimates further showed that only Butyricicoccus and Marvinbryantia presented stable negative associations with liver cirrhosis.No significant heterogeneity or horizontal pleiotropy was observed in the sensitivity analysis.Furthermore,the result of 16S rRNA sequencing also showed that healthy controls had a higher relative abundance of Butyricicoccus and Marvinbryantia than liver cirrhosis patients.Conclusions:Our study provides new causal evidence for the link between gut microbiota and liver cirrhosis,which may contribute to the discovery of novel strategies to prevent liver cirrhosis.

黄芪根瘤菌CA8561和JL84的部分16SrRNA基因序列的测定

报导了黄芪根瘤菌ＣＡ８５６１和ＪＬ８４的部分１６ｓｒＲＮＡ基因序列，测算了它们与已知根瘤菌、茎瘤菌各属模式种之间的遗传距离．结果表明，ＣＡ８５６１和ＪＬ８４均具有独立的进化路线，充分显示了黄芪根瘤菌的遗传多样性．

阿尔茨海默病患者肠道菌群改变与认知功能损伤的相关性

目的探讨阿尔茨海默病(Alzheimer′s disease,AD)患者肠道菌群改变及其与认知功能损伤相关性。方法根据临床痴呆评定量表(CDR),将35例AD患者分为轻中度组(n=15)和重度组(n=20),收集两组患者的粪便样本,对粪便标本进行16S rRNA高通量测序分析粪便中微生物群的组成和肠道菌群β多样性。采用调查问卷及量表调查AD患者的一般情况、抑郁状态及认知损伤程度。通过血液检测AD患者白细胞、甘油三酯、胆固醇、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、载脂蛋白A(ApoA)、载脂蛋白B(ApoB)、载脂蛋白E(ApoE)、脂蛋白a(Lpa)、血糖(Glu)、糖化血红蛋白(HbA1c),采用R软件进行粪便菌群与上述指标的相关性分析。结果AD重度组简易精神状态检查量表(MMSE)、重复性成套神经心理状态测验(RBANS)分值均低于AD轻中度组(P<0.01)。肠道菌群β多样性分析结果提示两组间菌群多样性差异有统计学意义(P<0.05)。重度组和轻中度组肠道菌门相对丰度及属相对丰度差异都有统计学意义(P<0.05)。AD患者大肠杆菌、志贺菌与Lpa呈正相关(P<0.01),与LDL呈负相关(P<0.05);双歧杆菌与RBANS、高脂血症呈正相关(P<0.05),与Lpa呈负相关(P<0.01);克雷伯杆菌与病程、CDR呈正相关(P<0.01);阿克曼菌与HLP呈负相关(P<0.05);肠球菌与CDR呈正相关(P<0.05);乳酸杆菌与年龄呈正相关(P<0.05);粪杆菌与CDR、病程、年龄和MMSE负相关(P<0.05)。结论AD患者伴随肠道菌群的改变,AD患者认知功能的损伤及其严重程度与肠道菌群多样性和相对丰度有关。

Changes in the gut microbiota of osteoporosis patients based on 16S rRNA gene sequencing:a systematic review and meta-analysis

Background:Osteoporosis(OP)has become a major public health issue,threatening the bone health of middle-aged and elderly people from all around the world.Changes in the gut microbiota(GM)are correlated with the maintenance of bone mass and bone quality.However,research results in this field remain highly controversial,and no systematic review or meta-analysis of the relationship between GM and OP has been conducted.This paper addresses this shortcoming,focusing on the difference in the GM abundance between OP patients and healthy controls based on previous 16S ribosomal RNA(rRNA)gene sequencing results,in order to provide new clinical reference information for future customized prevention and treatment options of OP.Methods:According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA),we comprehensively searched the databases of Pub Med,Web of Science,Embase,Cochrane Library,and China National Knowledge Infrastructure(CNKI).In addition,we applied the R programming language version 4.0.3 and Stata 15.1 software for data analysis.We also implemented the Newcastle-Ottawa Scale(NOS),funnel plot analysis,sensitivity analysis,Egger’s test,and Begg’s test to assess the risk of bias.Results:This research ultimately considered 12 studies,which included the fecal GM data of 2033 people(604 with OP and 1429 healthy controls).In the included research papers,it was observed that the relative abundance of Lactobacillus and Ruminococcus increased in the OP group,while the relative abundance for Bacteroides of Bacteroidetes increased(except for Ireland).Meanwhile,Firmicutes,Blautia,Alistipes,Megamonas,and Anaerostipes showed reduced relative abundance in Chinese studies.In the linear discriminant analysis Effect Size(LEfSe)analysis,certain bacteria showed statistically significant results consistently across different studies.Conclusions:This observational meta-analysis revealed that changes in the GM were correlated with OP,and variations in some advantageous GM might involve regional differences.