斯氏艾美耳球虫ITS1-5.8S rRNA-ITS2序列的克隆及PCR检测方法的建立

通过对多种鸡球虫和松鼠球虫18SrRNA和28SrRNA进行序列比对分析,在18SrRNA 3′端和28SrRNA 5′端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8SrRNA-ITS2序列,其大小为1 178bp,其中ITS1序列长度为423bp,5.8SrRNA为155bp,ITS2为600bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列相似性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。

基于ITS1-5.8S rRNA-ITS2序列兔肝球虫PCR检测方法的建立

通过对多种鸡球虫和松鼠球虫18S rRNA和28S rRNA进行序列比对分析,在18S rRNA 3'端和28S rRNA 5'端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8S rRNA-ITS2序列,其大小为1178bp,其中ITS1序列长度为423bp,5.8S rRNA为155 bp,ITS2为600 bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列同源性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。

Genetic relationship between Neobenedenia girellae and N. melleni inferred from 28S rRNA sequences

The fragments of 350 bp in 28S rRNA from the closely related monogenea of trematoda, Neobenedenia girellaeand N. melleni are obtained by polymerase chain reaction (PCR) amplified using a couple of special primers andthen sequenced. The results show that the comparison of 28S rRNA sequences, with only a base varying in 337bp accounting for 0.3% genetic difference, from the relative species N. girellae and N. melleni parasitized on thedifferent fishes in different farms displays that they possess a very high genetic similarity of 99.7%, higher thanthat of 99.41% for the single species N. melleni sampled in different areas, and the intraspecific divergence of N.melleni is 0.59%. Meanwhile, the interspecific differences between the two Neobenedenia and three Benedenia (i.e., B. lutjani, B. rohdei and B. seriolae) range from 2.08% to11.73%. In addition, UPGMA and MP molecularphylogenetic trees are constructed and proved to be consistent with each other. Though the morphologicalcharacteristics and the results of genetic diversity for the two Neobenedenia show a high similarity, whether theybelong to a single species or not are still undefined, and the more genes of them should be further investigated, incombination with the systematical and detailed morphological study.

大型海洋绿藻5.8S rRNA基因序列及系统发育分析

采用PCR方法,将扩增的产物直接测序,测定了孔石莼Ulva pertusa和浒苔Enteromorpha prolifera的5.8S rRNA基因序列,与肠浒苔、扁浒苔、缘管浒苔、礁膜、北极礁膜、袋礁膜和格氏礁膜等7种已知的大型海洋绿藻的同一基因序列合并进行比较,分析了核苷酸序列差异和碱基替换特点,并构建系统发生树。结果表明,浒苔和孔石莼的颠换率为0.690%,小于浒苔与肠浒苔、扁浒苔、缘管浒苔的颠换率;且浒苔与孔石莼之间的Kimura双参数距离也小于浒苔与肠浒苔、扁浒苔以及缘管浒苔之间的平均Kimura双参数距离。相对于浒苔属的其他物种,浒苔与孔石莼的亲缘关系更近。在构建的系统发生树中,缘管浒苔与孔石莼聚为一类,表现出与石莼属物种更近的亲缘关系。

广西扇棘单睾吸虫5.8S rRNA序列及二级结构的研究

目的比较分析广西人体扇棘单睾吸虫、扇棘单睾吸虫和钩棘单睾吸虫5.8SrRNA序列及二级结构,探讨他们的进化关系。方法提取广西扇棘单睾吸虫DNA,PCR扩增并测定5.8SrDNA序列;登录GenBank获取扇棘单睾吸虫和钩棘单睾吸虫5.8SrDNA序列。应用相关软件将5.8SrDNA基因转换成5.8SrRNA。采用Zuker算法,构建5.8SrRNA分子二级结构;计算3种吸虫间的遗传距离和同源性,分析5.8SrRNA序列及其二级结构特征。结果广西人体扇棘单睾吸虫与扇棘单睾吸虫5.8SrRNA分子序列比较显示第81、83、84、129和143位共5个碱基发生变异,这些突变碱基均位于5.8SrRNA分子二级结构未配对碱基区,未引起5.8SrRNA二级结构的明显变化;与钩棘单睾吸虫比较,显示第13、27、49、66、99、152、157位共7个碱基发生变异,其中3个碱基发生在配对区,4个替代发生在未配对区,碱基变异引起两种吸虫5.8SrRNA二级结构的明显不同;广西扇棘单睾吸虫与扇棘单睾吸虫和钩棘单睾吸虫的遗传距离分别为0.031和0.044,同源性分别为96.9%和95.6%。结论广西人体扇棘单睾吸虫5.8SrRNA序列与扇棘单睾吸虫高度同源,二级结构无明显不同,与钩棘单睾吸虫同源性较低,二级结构明显不同。

Effects of sepsis and its treatment measures on intestinal flora structure in critical care patients

BACKGROUND Sepsis is a common disease in intensive care units,with high morbidity and mortality.Intestinal microecology plays a vital part in the development and progression of this disease,possibly because sepsis and its treatment cause specific changes in the composition of the intestinal flora.AIM To investigate the characteristics of intestinal flora disturbance in sepsis patients treated with antibiotics.METHODS In this prospective comparative study,we enrolled ten patients with sepsis(sepsis group),hospitalized in the Department of Critical Care Medicine of the General Hospital,Ningxia Medical University,China(a class IIIa general hospital)from February 2017 to June 2017;ten patients without sepsis hospitalized in the same period(non-sepsis group)and ten healthy individuals(control group)were also enrolled.Fecal samples collected from the three groups were subjected to 16S rRNA gene sequencing and the intestinal flora diversity,structure,and composition were determined.Additionally,the dynamics of the intestinal flora diversity,structure,and composition in sepsis patients were investigated via 16S rRNA gene sequencing of samples collected 0 d,3 d,and 7 d after admittance to the intensive care unit.Correlations between the serum levels of procalcitonin,endotoxin,diamine oxidase,and D-lactic acid and the intestinal flora composition of sepsis patients were also investigated.RESULTS Compared with the healthy control group,sepsis and non-sepsis patients showed reduced intestinal floraα-diversity and a distinct flora structure,with Firmicutes as the dominant phylum,and significantly decreased proportions of Bacteroidetes,as well as Prevotella and Lachnospira,among other genera.Of note,the proportion of Enterococcus was significantly increased in the intestinal tract of sepsis patients.Interestingly,theα-diversity in the sepsis group decreased gradually,from days 1 to 7 of treatment.However,pairwise comparisons showed that both the diversity and structure of the intestinal flora were not significantly different considering the three different time points studied.Curiously,the serum levels of procalcitonin,endotoxin,diamine oxidase,and D-lactic acid in sepsis patients correlated with the prevalence of various bacterial genera.For example,the prevalence of Ruminococcus was positively correlated with serum procalcitonin,endotoxins,and diamine oxidase;similarly,the prevalence of Roseburia was positively correlated with serum procalcitonin,endotoxins,and D-lactic acid.CONCLUSION Sepsis patients in intensive care units show dysbiosis,lasting for at least 1 wk.

Fecal microbiota of three bactrian camels(Camelus ferus and Camelus bactrianus) in China by high throughput sequencing of the V3-V4 region of the 16S rRNA gene

This study aimed to reveal the microbial diversity in the fecal samples of bactrian camels using the 16 S r RNA sequencing analysis on the Illumina Mi Seq platform. Three fecal samples were collected from two geographical regions in China. Operational taxonomic unit（OTU） clustering was performed by identifying an OTU at 97% sequence identity. The alpha and beta diversities were applied to estimate the differences in microbial diversity among the three fecal samples. Totally, 4409, 3151 and 4075 OTUs in the fecal samples were identified in the Lop Nor wild camel（Camelus ferus）, the domestic camel（C. bactrianus） and Dunhuang wild camel（C. ferus）, respectively. The majority of bactreria were affiliated with phylum Firmicutes and Bacteroidetes in the three samples. The wild camels had higher gastrointestinal tract microbial diversity than the domestic one, while the microbial composition of the Lop Nor wild camel shared higher similarity with domestic camel at the genus and family levels than that of the Dunhuang wild camel did. Our results may provide a theoretical basis for assessing their health conditions and may thus be useful for protecting the critically endangered species of C. ferus.

16S rRNA Cloning and Identification of a Strain Isolated from Intestinal Tract of Rex Rabbit

This study was conducted to identify bacterial strain HeNan-001 isolated from intestinal tract of healthy rex rabbit. The strain was identified by gram staining, and OD600 values at different culture time were measured to develop a bacterial growth curve. The metabolic pathways of the bacterium using sugar in fermentation was identified with biochemical tubes. Total RNA of the strain was extracted, and total eDNA was obtained by Oligo （dT） method. Primers were designed using Primer 5.0 hip-software according to the 16S rRNA sequence published in GenBank, through cloning, ligation to vector PMD18T, construction of PMD18T/16S rRNA vector and transformation into DHSα, plasmid was extracted and sequenced, and the sequencing result was compared with sequences in NCBI followed by drawing of phylogenetic tree. The strain was verified to be double-stranded gram-positive cocci, which could ferment glucose, mannose, maltose and sorbitol, producing acids production, but failed to ferment sucrose, serum inulin and β-galactosidase. The phylogenetie tree analysis showed that the isolated bacterium shared the highest homology with an India isolate, and belongs to diplostreptococcus. This study provides significance experimental data and theoretical guidance for further development and utilization of the bacterial strain.

Vertical microbial profiling of water column reveals prokaryotic communities and distribution features of Antarctic Peninsula

Prokaryotic diversity and community composition in the water column of eight stations(63 samples) around the Antarctic Peninsula of the Southern Ocean were investigated. Through pyrosequencing of the V3–V4hypervariable regions of the 16S ribosomal RNA gene, we characterized 4 720 089 valid reads representing 48 188operational taxonomic units(OTUs, 97% similarity). The community was dominated by the phyla Pseudomonadota(original name: Proteobacteria, 47%), Oxyphotobacteria(26%), and Bacteroidota(original name: Bacteroidetes, 18%), which comprised an average of 91% of the total OTUs in all samples. The prokaryotic community composition varied vertically within the water column. Water column prokaryotic communities exhibited a clear depth profile, with higher microbial richness and higher diversity observed with increasing water depth. Cluster analysis of the community composition of water column samples exhibited a similar trend with depth. Correlation with environmental factors suggested distinct variation in prokaryotic community composition with changes in depth, salinity, temperature and dissolved oxygen levels. Functional prediction showed presence of active nitrogen, sulphur and methane metabolic cycles along the vertical transect of the studied region. These results will improve our knowledge of prokaryotic diversity and community composition at different depth of water column for better understanding of the microbial ecology and nutrient cycles in Antarctic Peninsula region of the Southern Ocean.

Sarcopenia and gut microbiota alterations in patients with hematological diseases before and after hematopoietic stem cell transplantation

Objective: The aim of this study was to investigate the prevalence of sarcopenia(SP) and its relationship with gut microbiota alterations in patients with hematological diseases before and after hematopoietic stem cell transplantation(HSCT).Methods: A total of 108 patients with various hematological disorders were selected from Peking University People’s Hospital. SP was screened and diagnosed based on the 2019 Asian Sarcopenia Diagnosis Strategy. Physical measurements and fecal samples were collected, and 16S rRNA gene sequencing was conducted. Alpha and beta diversity analyses were performed to evaluate gut microbiota composition and diversity.Results: After HSCT, significant decreases in calf circumference and body mass index(BMI) were observed,accompanied by a decline in physical function. Gut microbiota analyses revealed significant differences in the relative abundance of Enterococcus, Bacteroides, Blautia and Dorea species before and after HSCT(P<0.05). Before HSCT, sarcopenic patients had lower Dorea levels and higher Phascolarctobacterium levels than non-sarcopenia patients(P<0.01). After HSCT, no significant differences in species abundance were observed. Alpha diversity analysis showed significant differences in species diversity among the groups, with the highest diversity in the postHSCT 90-day group and the lowest in the post-HSCT 30-day group. Beta diversity analysis revealed significant differences in species composition between pre-and post-HSCT time points but not between SP groups. Linear discriminant analysis effect size(LEfSe) identified Alistipes, Rikenellaceae, Alistipes putredinis, Prevotellaceae defectiva and Blautia coccoides as biomarkers for the pre-HSCT sarcopenia group. Functional predictions showed significant differences in anaerobic, biofilm-forming and oxidative stress-tolerant functions among the groups(P<0.05).Conclusions: This study demonstrated a significant decline in physical function after HSCT and identified potential gut microbiota biomarkers and functional alterations associated with SP in patients with hematological disorders. Further research is needed to explore the underlying mechanisms and potential therapeutic targets.

Prokaryotic diversity and community composition in the surface sediments of the Changjiang River Estuary in summer

Microorganisms are fundamental for the functioning of marine ecosystems and are involved in the decomposition of organic matter, transformation of nutrients and circulation of biologically-important chemicals. Based on the complexity of the natural geographic characteristics of the Changjiang River Estuary, the geographic distribution of sedimentary microorganisms and the causes of this distribution are largely unexplored. In this work, the surface sediment samples from the adjacent sea area of the Changjiang River Estuary were collected. Their prokaryotic diversity was examined by high-throughput sequencing technology, and the environmental factors of the bacterial community were investigated. The results indicated that the distribution of prokaryotic communities in the sediments of the study areas showed obvious spatial heterogeneity. The sampling sequences divided the sample regions into three distinct clusters. Each geographic region had a unique community structure, although Proteobacteria, Bacteroidota, Desulfobacterota, Acidobacteriota, and Actinobacteriota all existed in these three branches. Canonical correspondence analysis demonstrated that prokaryotic diversity and community distribution were significantly correlated with the geographic location of sediment, seawater depth, and in particular, nutrient content(e.g., total phosphorus, total organic carbon and dissolved oxygen). Moreover, it was found for the first time that the metal ions obviously affected the composition and distribution of the prokaryotic community in this area. In general, this work provides new insights into the structural characteristics and driving factors of prokaryotic communities under the background of the ever-changing Changjiang River Estuary.

Identification of Bacterial Fish Pathogens in Brazil by Direct Colony PCR and 16S rRNA Gene Sequencing

Intensive fish farming systems in Brazil have increased the disease incidence, mainly of bacterial origin, due to higher stocking density, high organic matter levels and poor quality of the aquatic environment that causes high mortality rates during outbreaks. The identification of pathogenic species using a fast and reliable method of diagnosis is essential for successful epidemiological studies and disease control. The present study evaluated the use of direct colony PCR in combination with 16S rRNA gene sequencing to diagnose fish bacterial diseases, with the goal of reducing the costs and time necessary for bacterial identification. The method was successful for all 178 isolates tested and produced bands with the same intensity as the standard PCR performed using pure DNA. In conclusion, the genetics methods allowed detecting the most common and important pathogens in Aquaculture, including 12 species of occurrence in Brazilian fish farms. The results of the present study constitute an advance in the available diagnostic methods for bacterial pathogens in fish farms.

RETRACTED:Effect of Long-Term Inorganic Fertilization on Diversity and Abundance of Bacterial and Archaeal Communities at Tillage in Irrigated Rice Field

Short Retraction Notice The paper does not meet the standards of "Advances in Bioscience and Biotechnology". This article has been retracted to straighten the academic record. In making this decision the Editorial Board follows COPE's Retraction Guidelines. The aim is to promote the circulation of scientific research by offering an ideal research publication platform with due consideration of internationally accepted standards on publication ethics. The Editorial Board would like to extend its sincere apologies for any inconvenience this retraction may have caused. Editor guiding this retraction: Prof. Abass Alavi (EiC of ABB). Please see the article page for more details. The full retraction notice in PDF is preceding the original paper which is marked "RETRACTED".

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.

异源四倍体鲫鲤、三倍体湘云鲫和它们父母本线粒体DNA12SrRNA基因遗传变异的分析

采用质粒克隆测序方法 ,获得了异源四倍体鲫鲤 5个个体、异源四倍体鲫鲤雌核发育二倍体后代 2个个体、三倍体湘云鲫 2个个体及红鲫、湘江野鲤和日本白鲫各 1个个体的线粒体DNA 12SrRNA基因的全序列。经对比发现 ,异源四倍体 5个个体共享 2种单元型 ,异源四倍体鲫鲤雌核发育二倍体后代 2个个体、三倍体湘云鲫 2个个体以及红鲫、湘江野鲤和日本白鲫各 1个个体分别共享 1种单元型。用MEGA 1 0软件分析了它们的碱基组成和核苷酸序列差异 ,用邻接法构建系统进化树。它们间的序列同源性在 95 %～ 99%之间 ,异源四倍体鲫鲤、三倍体湘云鲫和它们母本 (分别为红鲫和日本白鲫 )之间的序列同源性大于异源四倍体鲫鲤、三倍体湘云鲫和它们父本(分别为湘江野鲤和异源四倍体鲫鲤 )之间的序列同源性 ,结果表明 :异源四倍体鲫鲤和三倍体湘云鲫在线粒体DNA 12SrRNA基因上具有母性遗传特征。另一值得注意的地方是异源四倍体鲫鲤经过 9代 (F3 F11)繁殖后 ,在 5个个体中发现了 2种单元型 ,说明在四倍体基因库中存在遗传多样性 ,为四倍体基因库的繁殖。

2种三疣梭子蟹居群线粒体Cyt b和S-rRNA基因片段序列变异研究

目的:通过对2种三疣梭子蟹居群样线粒体Cyt b和S-rRNA基因片段的序列分析,探讨其遗传变异,为种质资源的管理、保存和利用提供依据.方法:采集山东潍坊和福建厦门两个不同居群的三疣梭子蟹样,提取其线粒体DNA.由Genebank获得三疣梭子蟹完整线粒体DNA序列(登陆号:NC_005037),设计扩增Cyt b和S-rRNA基因片段引物,经PCR扩增获得预期产物,纯化后测序.结果:以提取的线粒体DNA为模板扩增出特异性强的Cyt b和S-rRNA基因片段,与预期条带长度429 bp和465 bp吻合;2个基因片段的AT含量均高于GC含量;Cyt b基因片段有3个位点发生了突变,S-rRNA只有1个位点发生了突变;在两种居群的4个突变位点中,2个位置突变为相同的碱基,且所有突变位点均呈转换方式.结论:Cyt b基因序列比S-rRNA基因序列具有相对高的突变率;研究的2个基因片段的突变位点的碱基置换主要呈转换方式;来自2个三疣梭子蟹居群样本的线粒体S-rRNA基因的突变发生在同一位点且突变为相同碱基,表明2个居群具有较近的亲缘关系.

16SrRNA序列分析法在大气微生物检测中的应用

随着微生物核糖体数据库的日益完善 ,16SrRNA序列分析技术已应用于海洋、湖泊和土壤等环境微生物多样性的分析 ,但尚未见其在大气微生物菌群分析中的应用报道。本研究选择 5株大气中采集分离的菌株 ,通过细菌 16SrRNA通用引物PCR扩增其对应序列 ,直接对PCR产物进行测序 ,分析鉴定其对应细菌的种属 ,并将该结果同细菌表型鉴定、全自动微生物分析仪以及气相色谱分析结果加以比较。结果表明 16SrRNA序列分析获得的鉴定结果与表型分析和气相色谱分析结果较为一致 ,该方法具有快速、准确和不依赖于细菌生长状态等优点 ,说明 16SrRNA序列分析法可以作为大气微生物分析的一个有效技术。

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。

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。

用18S rRNA序列探讨8种绿藻分子系统发育

测定了宽礁膜Monostroma latissimum、浒苔Enteromorpha prolifera、孔石莼Ulva pertusa的18S rRNA基因序列,并与肠浒苔E.intestinalis、穿孔石莼U.fenesrtata、石莼U.lactuca、礁膜M.nitidum和格氏礁膜Monostroma grevillei5种已知的大型海洋绿藻的同一基因序列合并进行比较,分析了核苷酸序列差异和碱基替换特点,并构建系统发生树。结果表明:浒苔与肠浒苔之间的Kimura双参数距离大于浒苔与石莼属3个物种之间的Kimura双参数距离;用UPGMA法和MP法构建的系统树基本一致,在构建的系统发生树中,浒苔与石莼属物种聚为一类,表现出与石莼属物种更近的亲缘关系。