PCR指纹图谱技术分析人体口腔内微生物菌群结构

目的 应用PCR-DGGE指纹图谱技术对人体口腔微生物菌群结构进行系统性研究.方法 对1例健康人唾液周期性采集的样品和8例健康人个体的唾液与牙菌斑采集的样品,进行微生物群落总DNA的抽提.以此为模板扩增16S rRNA V3可变区,产物经DGGE指纹图谱分析其组成结构,并运用UVIBAND/MAP等软件比较所得群落指纹图谱的相似性指数.结果 同一健康人个体不同采样时间的唾液菌群结构相似性系数＞74%,通过对不同健康个体口腔样本的研究,发现同一个体的唾液与牙菌斑菌群结构存在差异（84%～95%）.结论 同一健康个体其唾液微生物菌群在一定时间内基本稳定,仅有微小的变化;唾液与同个体牙菌斑的微生物组成虽然存在差异,但这种差异要明显小于个体间的差异.     

Molecular diversity analysis of rumen methanogenic Archaea from goat in eastern China by DGGE methods using different primer pairs

Aims:  To screen a pair of primers suitable for denaturing gradient gel electrophoretic (DGGE) analysis of ruminal methanogenic Archaea and to detect the archaeal communities in the rumen of goat.
Methods and Results:  Nine primer pairs for 16S rDNA of methanogenic Archaea , including six for directed polymerase chain reaction (PCR) and three for nested PCR were first evaluated by PCR amplification of the total DNA from rumen fluids and bacteria. The DGGE analysis of rumen fluids was then conducted with three primer sets (344fGC/915r, 1106fGC/1378r and 519f/915rGC) of the nine pairs tested. Good separation and quality of patterns were obtained in DGGE analysis with primer pairs 1106fGC/1378r and 519f/915rGC. A total of 40 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic Archaea while primer pair 519f/915rGC had better amplification ranges than the other two primer pairs.
Conclusions:  The procedure of DGGE analysis with primer pair 519f/915rGC was more suitable for investigating methanogenic archaeal community in the rumen. The dominant methanogenic Archaea in the rumen of goat was Methanobrevibacter sp. and an unidentified methanogenic Archaea .
Significance and Impact of the Study:  One pair of primers suitable for DGGE analysis of ruminal methanogenic Archaea was obtained and the molecular diversity of ruminal methanogenic Archaea in goat was investigated by PCR-DGGE.     

我国主要生态区域绿豆慢生根瘤菌的遗传多样性和系统发育研究

利用16SrRNAPCR-RFLP、16SrRNA序列分析以及16S-23SrRNAIGS(IntergeneticSpacer)PCR-RFLP技术对分离自中国主要生态区域的44株慢生型绿豆根瘤菌和5株参比菌株进行了遗传多样性和系统发育研究。16SrRNAPCR-RFLP分析表明:在76%的相似水平上,所有供试菌株可分为三大类群:群I由LYG1等13株慢生根瘤菌组成,该群在系统发育上与B.japonicum和B.liaoningense的参比菌株存在一定的差异;群Ⅱ由XJ1等21株供试菌株、B.japonicum和B.liaoningense的代表菌株组成;群Ⅲ由10株来自广东和广西的菌株和B.elkanii的代表菌株组成。16S-23SrRNAIGSPCR-RFLP分析将供试菌株分为A、B两大群。群A由34株供试菌株、B.japonicum和B.liaoningense的代表菌株组成。在85%的相似性水平上,可再分为AⅠ、AⅡ和AⅢ3个亚群。群B由10株分离自广西和广东的菌株和B.elkanii的代表菌株组成。在85%的相似性水平上,可再分为BI和BⅡ两亚群,表现出一定的多样性。与16SrRNAPCR-RFLP相比,16S-23SrRNAIGSPCR-RFLP具有更高的解析度,供试菌株表现出更加丰富的遗传多样性。分离自中国新疆、广东和广西等地的菌株在分群上具有较为明显的地域特征。     

种植体周围炎生物膜的微生物群落多样性研究进展

种植体周围炎是发生在骨性结合种植体周围组织的炎症,是由微生物引发的感染性疾病,可引起种植体周围支持组织丧失而导致种植失败。阐明种植体周围炎生物膜的微生物学基础,可为制定相应防治策略提供理论依据。随着测序技术的发展,基于16S rRNA基因的测序分析技术逐渐应用于与口腔种植体相关的微生物学研究,使人们对种植体周围炎生物膜的微生物群落多样性有了更全面的了解,也进一步认识到种植体周围炎和牙周炎菌斑生物膜的微生物结构存在显著差别。本文根据基于16S rRN基因A序列分析技术的最新研究成果,对种植体周围炎菌斑生物膜的微生物学研究进展作一综述。     

Genomic and phenotypic heterogeneity of Acidithiobacillus spp. strains isolated from diverse habitats in China

The genetic variability among 32 Chinese Acidithiobacillus spp. environmental isolates and four reference strains representing three recognized species of the genus Acidithiobacillus was characterized by using a combination of molecular methods, namely restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers, repetitive element PCR, arbitrarily primed PCR and 16S rRNA gene sequence analyses. 16S rRNA gene sequences revealed that all Acidithiobacillus spp. strains could be assigned to seven groups, three of which encompassed the Acidithiobacillus ferrooxidans strains from various parts of the world. A comparative analysis of the phylogenetic Group 1 and 2 was undertaken. Restriction fragment length polymorphism results allowed us to separate the 35 Acidithiobacillus strains into 15 different genotypes. An integrated phenotypic and genotypic analysis indicated that the distribution of A. ferrooxidans strains among the physiological groups were in agreement with their distribution among the genomic groups, and that no clear correlation was found between the genetic polymorphism of the Acidithiobacillus spp. strains and either the geographic location or type of habitats from which the strains were isolated. In addition, five unidentified sulfur-oxidizing isolates may represent one or two novel species of the genus Acidithiobacillus. The results showed that the Chinese Acidithiobacillus spp. isolates exhibited a high degree of genomic and phenotypic heterogeneity.     

16s rRNA基因克隆文库分析比较牙周炎患者和健康人口腔唾液微生物多样性

【目的】通过对同一地区、同一民族牙周炎患者和健康人的唾液微生物群落结构的分析,探寻牙周炎患者口腔微生物的多样性。【方法】采集甘肃东乡族自治县的东乡族牙周炎患者和健康人唾液各5例,分别记作DP(东乡牙周)和DH(东乡健康),提取细菌总DNA,构建16S r RNA基因克隆文库,测序后利用MOTHUR、MEGA 4.0、Clustal X 3.0等软件对测序结果进行分析。【结果】所有样本共检测出115个OTUs(DP 60,DH 75),归属于6个门,27个属。TM7是DP组特有的优势菌门。仅在DP组中检测到的优势菌属是梭菌属(Fusobacterium)、卟啉单胞菌属(Porphyromonas)、消化链球菌属(Peptostreptococcus)和TM7_genera。【结论】发现牙周炎患者与健康人口腔唾液微生物存在一定差异。其中,TM7、梭菌属和消化链球菌属在牙周病中的作用值得进一步研究。     

5株北极微藻藻际环境的细菌多样性

对5株北极微藻,如脆杆藻(Fragilariopsis sp.)、微单胞藻(Micromonas sp.)、四棘藻(Attheya septentrionalis)、海链藻(Thalassiosira sp.)和小球藻(Chlorella sp.)的不同生长时期的粘附细菌和游离细菌的16S rRNA基因进行PCR-DGGE分析,研究藻际环境的细菌多样性。结果表明,5株微藻具有不同的藻际微生物群落结构组成,其中微单胞藻、脆杆藻、四棘藻和海链藻的藻际细菌主要由Cyanobacteria(藻蓝细菌)、α-Proteobacteria(α-变形菌纲)和γ-Proteobacteria(γ-变形菌纲)组成,仅微单胞藻和脆杆藻检测出CFB(Cytophaga-Flexibacter-Bacteroides,噬纤维菌-屈挠杆菌-拟杆菌)。小球藻由Cyanobacteria、CFB、α-Proteobacteria和β-Proteobacteria(β-变形菌纲)组成。微单胞藻的藻际菌群结构稳定,不同生长时期的游离细菌和粘附细菌组成差异不明显。3株硅藻-脆杆藻、四棘藻和海链藻的游离细菌主要由γ-Proteobacteria组成,小球藻的游离细菌主要为β-Proteobacteria,而5株微藻的粘附细菌主要由Cyanobacteria组成。从DGGE图谱来看,在脆杆藻生长的延滞期、指数期和稳定期,其藻际游离细菌和粘附细菌的16S rRNA基因扩增条带数量和位置均有明显差异,但优势扩增条带较稳定;其他4株藻粘附细菌和游离细菌的扩增条带比较稳定,说明藻际关联菌群结构较稳定。藻菌种间特异性关系为不同微藻藻株提供了重要的线索,同时也带来更多的隐藏在藻际环境中的信息。     

Changes in the diversity of pig ileal lactobacilli around weaning determined by means of 16S rRNA gene amplification and denaturing gradient gel electrophoresis

Our study aimed to provide a comprehensive characterization of changes in porcine intestinal Lactobacillus populations around the time of weaning based on 16S rRNA gene amplification and denaturing gradient gel electrophoresis (DGGE). DNA was extracted from the ileal contents of piglets at weaning (28 days of age) and after 1, 2, 5 and 11 days. PCR amplicons (V2-V3 fragments of 16S rRNA genes) were separated using DGGE. Predominant bands were excised and sequenced after reamplification. A band corresponding to Lactobacillus salivarius was present 1 and 2 days post-weaning (pw), while Lactobacillus crispatus was detected only 1 and 11 days pw. Lactobacillus sobrius gave the most dominant band in all animals. The number of bands decreased from 13+/-3 at weaning to 9+/-1 at 5 days pw, but the species richness had recovered by 11 days pw. The similarity of profiles between sampling days was high for 1 and 2 days pw (>91%), but was low for 5 and 11 days pw (<59%). The diversity of the profiles was lower 5 days pw, based on the Shannon diversity index (0.83+/-0.076 vs. 1.02+/-0.127 at weaning, P=0.042), but had recovered to preweaning values by 11 days pw. The application of group-specific DGGE showed that the Lactobacillus community within the porcine ileum undergoes dramatic, partly reversible changes as a consequence of weaning.     

广西水牛瘤胃中的细菌多样性

[目的]了解广西水牛瘤胃中细菌的组成及其可能的降解纤维素细菌的主要类群。[方法]提取水牛瘤胃内容物和高效降解滤纸的水牛瘤胃内容物的富集培养物的宏基因组DNA,以宏基因组DNA为模板,扩增16S rRNA基因序列,构建该两种样品的细菌的16S rRNA基因文库。通过对16S rRNA基因序列的分析,了解这两种样品的细菌群体种类及数量。 [结果] 水牛瘤胃内容物与其富集培养物中均主要含有LGCGPB (low G+C Gram-Positive Bacteria)、CFB (Cytophaga-Flexibacter-Bacteroides)两大类菌群和少数的螺旋体菌（Spirochaetes）,且LGCGPB所占的比例都是最高的,LGCGPB在水牛瘤胃内容物细菌中的比例为56.66%,而在富集培养物细菌中的比例升高为73.33%。在水牛瘤胃内容物中,丝状杆菌（Fibrobacteres）占3.33%,但在富集培养物中未被检测到。而在富集培养物中占13.33%的变形杆菌（Proteobacteria）,在水牛瘤胃内容物中未被检测到。本研究还发现了分类地位尚未明确的一菌群（R46）。[结论]细菌类群LGCGPB、Proteobacteria可能在水牛瘤胃中的纤维素降解过程中起重要作用。此外,水牛瘤胃中的细菌组成和牦牛、牛、羊瘤胃中的细菌组成较相似但比例有所不同。     

PCR-DGGE对长江河口八种野生鱼类肠道菌群多样性的比较研究

目的分析长江河口捕获的8种野生鱼类的肠道菌群多样性的差异并观察这种差异与食性的联系。方法采用PCR-DGGE(denaturing gradient gel electrophoresis)技术,DGGE图谱用PCA(principal component analy-sis)方法进行分析。结果建立了长江口8种鱼野生条件下肠道菌群的DGGE指纹图谱,观察到它们在野生条件下的肠道菌群的差异。其中,营底栖生活的舌鰕虎鱼的肠道菌群和其他7种野生鱼有着明显的差异,其他7种鱼的肠道菌群多样性的差异与它们的食性差异相关。结论PCR-DGGE技术是一种能够快速有效地分析研究鱼类肠道菌群结构的技术。8种野生鱼的肠道菌群的结构有明显的差别,并且食性差异大的鱼类之间肠道菌群差异也     

我国南北大豆产区慢生大豆根瘤菌的遗传多样性和系统发育研究

利用16S rRNA基因RFLP、16S rRNA基因序列分析以及16S-23S rRNA IGS PCR RFLP技术对分离自我国南北大豆产区的慢生大豆根瘤菌进行了群体遗传多样性和系统发育研究。16S rRNA基因PCR RFLP分析以及16S rRNA基因序列分析结果表明:所有供试慢生大豆根瘤菌可分为B.japonicum和B.elkanii两个类群,其中属于B.japonicum的为优势种群,占供试菌株的91%,属于B.elkanii的仅占9%,多样性水平较低。16S-23S rRNA IGS PCRRFLP研究结果表明:属于B.japonicum的慢生根瘤菌具有较丰富的遗传多样性,在69%的相似性水平上可分为群Ⅰ和群Ⅱ两大类群。群I的菌株以分离自黑龙江和河北等北部区域的菌株为代表,群Ⅱ的菌株以分离自广西和江苏等南部地域的菌株为代表,反映出明显的地域特征。两群菌株在系统发育上均与USDA6、USDA110和USDA122等B.japonicum的模式或代表菌株有差异。     

Actinobacterial diversity from marine sediments collected in Mexico

Seventeen different media known to support the growth and isolation of members of the class Actinobacteria were evaluated as selective isolation media for the recovery of this microbial group from marine sediments samples collected in the Gulf of California and the Gulf of Mexico. A general selective isolation procedure was employed for six sediments and nearly 300 actinomycetes were recovered from the selective isolation plates. Full 16S rRNA gene sequencing revealed that the isolates belonged to several actinobacterial taxa, notably to the genera Actinomadura, Dietzia, Gordonia, Micromonospora, Nonomuraea, Rhodococcus, Saccharomonospora, Saccharopolyspora, Salinispora, Streptomyces, “Solwaraspora” and Verrucosispora. Previous works on marine sediments have been restricted to the isolation of members of the genera Micromonospora, Rhodococcus and Streptomyces. This study provides further evidence that Actinobacteria present in marine habitats are not restricted to the Micromonospora-Rhodococcus-Streptomyces grouping. Indeed, this first systematic study shows the extent of actinobacterial diversity that can be found in marine sediments collected in Mexico and probably, worldwide. The 16S rRNA gene sequences of marine isolates A1, AA2, AA6, AB1, AB2, AG1, AI2, AK1, AL2, AO1, AO3, AR1, AW1, B1, BB1, BC1, C5, R1, R2, R3, AV1, AE1, AI1, AN1 and AP1 determined in this study have been deposited under GenBank accession numbers EU714241–EU714258 and FJ462359–FJ462365, respectively.     

现代分子生物学技术在冰川微生物生态研究中的应用

研究冰川中微生物的多样性对于揭示环境气候变迁,研究生物进化,开发微生物资源具有重要意义,现代分子生物学的发展为研究冰川微生物的多样性提供了行之有效的方法.简要综述了16S rDNA文库构建、变性梯度凝胶电泳、限制性片段长度多态性和荧光原位杂交等技术的原理及在冰川微生物生态研究中的应用现状.     

Influence of petroleum contamination and biostimulation treatment on the diversity of Pseudomonas spp. in soil microcosms as evaluated by 16S rRNA based-PCR and DGGE

AIMS: The aim of this study was to apply a group specific PCR system followed by denaturing gradient gel electrophoresis (DGGE) analysis to evaluate the effect of oil contamination and the biostimulation process on the diversity of Pseudomonas populations in soil ecosystems. METHODS AND RESULTS: Direct DNA extraction from biostimulated- and oil-contaminated soil samples was performed. Primers specific for the genus Pseudomonas spp. were used to amplify 16S rRNA genes and then a semi-nested PCR reaction was applied to obtain smaller fragments for comparing the PCR products by DGGE. Whether in bulk, oil-contaminated or biostimulated soils, the DGGE profiles revealed little change in Pseudomonas community throughout the 270 days of experiment. The presence of a few additional bands observed only in treated samples indicated that a bacterial shift occurred with the addition of nutrients and with oil contamination. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of semi-nested PCR and DGGE was found to be a rapid and sensitive technique to study the diversity within the genus Pseudomonas and may be suitable for further studies concerning the role of this bacterial group in large-scale oil-contaminated areas.     

茶园土壤微生物群落基因多样性

应用PCR技术,直接从土壤中抽提总DNA,扩增16S rDNA V3 片段,采用变性梯度凝胶电泳(DGGE)分析16S rDNA V3 片段的多态性,研究了杭州西湖梅家坞不同植茶年龄(8、50和90年)、不同利用方式(茶园、荒地和林地)的土壤微生物群落基因多样性.结果表明：不同植茶年龄和不同土地利用方式影响土壤微生物群落的基因多样性.荒地、茶园和林地土壤微生物群落基因多样性指数明显不同(P<0.05),其排列顺序为荒地>茶园>林地.不同植茶年龄的土壤中,50年茶园土壤的微生物群落基因多样性指数、微生物量碳和基础呼吸明显高于8年和90年茶园土壤(P<0.05).     

新疆两典型微咸水湖水体免培养古菌多样性

微咸水湖是湖泊演化过程中的一个重要中间状态,以新疆两典型微咸水湖-赛里木湖和柴窝堡湖水为研究对象,采用微孔滤膜收集菌体,SDS-酚-氯仿抽提法直接提取湖水总DNA,利用古菌16S rRNA基因通用引物进行PCR扩增,分别构建两湖古菌16S rRNA基因克隆文库。用限制性内切酶Hae Ⅲ对随机挑选的阳性克隆子进行酶切分型,分别得到7个和8个可操作分类单元(Operational Taxonomic Units, OTUs),两文库覆盖率均大于98%。BLAST比对和系统发育分析表明赛里木湖全部克隆子归属于泉古菌门(Crenarchaeota),97%的克隆子与不同环境免培养氨氧化泉古菌有较高的序列相似性(>97%)。柴窝堡湖水古菌归为3个门:Thaumarchaeota (81.2%)、广古菌门(Euryarchaeota)(13%)和泉古菌门(Crenarchaeota) (5.8%),81.2%的克隆子与具有氮代谢功能的氨氧化古菌纯培养物具有较高的序列相似性(97%-98%),13%的克隆子与已分离到的产甲烷古菌序列同源性大于97%。研究发现新疆微咸水湖可能存有大量新划分的古菌Thaumarchaeota门类群、可培养氨氧化及产甲烷古菌类群,两典型微咸水湖泊中古菌类群多样性较低且群落组成差异大。     

A comparison of DNA- and RNA-based clone libraries from the same marine bacterioplankton community

Clones from the same marine bacterioplankton community were sequenced, 100 clones based on DNA (16S rRNA genes) and 100 clones based on RNA (16S rRNA). This bacterioplankton community was dominated by alpha-Proteobacteria in terms of repetitive DNA clones (52%), but gamma-Proteobacteria dominated in terms of repetitive RNA clones (44%). The combined analysis led to a characterization of phylotypes otherwise uncharacterized if only the DNA or RNA libraries would have been analyzed alone. Of the DNA clones, 25.5% were found only in this library and no close relatives were detected in the RNA library. For clones from the RNA library, 21.5% of RNA clones did not indicate close relatives in the DNA library. Based on the comparisons between DNA and RNA libraries, our data indicate that the characterization of the bacterial community based on RNA has the potential to characterize distinct phylotypes from the marine environment, which remain undetected on the DNA level.     

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities

A protocol was devised which permitted the extraction of DNA from deep marine sediments up to 503 m below the sea floor. These sediments have been laid down over the last 3 million years. 16S rRNA gene sequences were amplified from the DNA by the polymerase chain reaction. The details of the successful extraction and polymerase chain reaction methodology varied between samples from different depths. This emphasizes the attention to detail required to allow the diversity of bacteria in these deep sediments to be studied.     

Assessing the diversity of bacterial communities from marine sponges and their bioactive compounds

Symbiotic bacteria play vital roles in the survival and health of marine sponges. Sponges harbor rich, diverse and species-specific microbial communities. Symbiotic marine bacteria have increasingly been reported as promising source of bioactive compounds. A culturomics-based study was undertaken to study the diversity of bacteria from marine sponges and their antimicrobial potential. We have collected three sponge samples i.e. Acanthaster carteri, Rhytisma fulvum (soft coral) and Haliclona caerulea from north region (Obhur) of Red Sea, Jeddah Saudi Arabia. Total of 144 bacterial strains were isolated from three marine sponges using culture dependent method. Screening of isolated strains showed only 37 (26%) isolates as antagonists against oomycetes pathogens (P. ultimum and P. capsici). Among 37 antagonistic bacteria, only 19 bacterial strains exhibited antibacterial activity against human pathogens (Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 29212). Four major classes of bacteria i.e γ-Proteobacteria, α-Proteobacteria, Firmicutes and Actinobacteria were recorded from three marine sponges where γ-Proteobacteria was dominant class. One potential bacterial strain Halomonas sp. EA423 was selected for identification of bioactive metabolites using GC and LC-MS analyses. Bioactive compounds Sulfamerazine, Metronidazole-OH and Ibuprofen are detected from culture extract of strain Halomonas sp. EA423. Overall, this study gives insight into composition and diversity of antagonistic bacterial community of marine sponges and coral from Red Sea and presence of active metabolites from potential strain. Our results showed that these diverse and potential bacterial communities further need to be studied to exploit their biotechnological significance.