舌苔细菌PCR-DGGE分析条件的建立与优化

目的针对口腔舌苔细菌16S rDNA序列进行变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)适用序列的筛选及电泳条件的优化。方法以DGGE图谱的高分辨率为指标,选择舌苔细菌DGGE分离最适的16S rDNA高变区、电泳电压和电泳时间,并采用优化的条件分析健康青年人舌苔细菌群落的分布。结果舌苔细菌16S rDNA V3高变区引物序列能获得更加丰富清晰的DGGE条带;基于该区,当变性剂浓度为30%～60%、电泳温度60℃、电压60 V和电泳时间14 h时能得到分辨率最佳的DGGE图谱。运用此优化条件对12个样本舌苔细菌群落的分析表明,舌苔微生物主要由厚壁菌门、梭杆菌门、拟杆菌门和变形菌门等组成。优化后的DGGE技术对舌苔细菌多样性的分析具有准确性、灵敏性和可重复性。结论 DGGE图谱显示,不同分析条件对图谱类型和细菌多样性指数均有所差异。利用优化的DGGE条件能有效分离舌苔细菌16S rDNA V3区序列,为口腔微生物群落结构分析提供可靠的技术支持,也为其他不同生态细菌的多样性分析提供参考。     

新疆一号冰川土壤细菌多样性的研究*

应用变性梯度凝胶电泳(DGGE)技术分离PCR扩增的16S rDNA来研究土壤微生物的多样性。直接从新疆一号冰川不同海拔高度的土壤样品中提取总DNA。用两套细菌通用引物分别扩增16S rDNA的V3和V6/V9高变区的特异性片段,PCR产物进行DGGE分析。PCR-DGGE图谱表明,PCR产物经DGGE检测到的条带清晰且分离效果好。结果表明,PCR-DGGE是一种快速研究微生物群落结构的有效方法。     

PCR-DGGE法在慢性胃炎病人舌苔菌群结构研究中的应用

在早间未刷牙和进食的情况下,刮取胃炎病人与正常人舌苔,去除杂质,分离菌体,采用酚/氯仿法抽提细菌基因组DNA,并对其中16S rDNA V3可变区进行聚合酶链式反应(PCR)扩增和变性梯度凝胶电泳(DGGE)测定.用Bionumerics软件对DGGE分子指纹图谱进行舌苔菌群结构相似性分析.实验结果表明,采用该方法成功地扩增出16S rDNA V3区片段,为230 bp.DGGE分子指纹图谱结果表明,正常人的舌苔菌群最高相似性为0.74,胃炎病人的舌苔菌群的最高相似性为0.52,正常人的舌苔菌群与胃炎病人的舌苔菌群相似性最高为0.38,即胃炎病人舌苔菌群结构发生了变化.     

云南腾冲热海两热泉菌藻席细菌多样性的研究

应用显微形态观察和变性梯度凝胶电泳(DGGE)对云南腾冲热海两热泉菌藻席的细菌多样性进行了比较分析.直接从环境样品中提取总DNA,用两套细菌通用引物进行PCR扩增,得到包含V8和V9高变区的16S rDNA片段,进行DGGE分析,结合形态观察,结果显示,热泉菌藻席中存在丰富的细菌多样性,且不同温度范围的菌藻席细菌组成差异显著.     

应用变性梯度凝胶电泳和16S rDNA序列分析对kefir粒中细菌多样性的研究

以开菲尔(Kefir)粒为材料,经过DNA抽提和16S rDNA V3区PCR扩增,扩增产物经变性梯度凝胶电泳(DGGE)分离并切割电泳条带进行序列测定,并与现有的数据库进行了比较,对Kefir粒的细菌多样性进行分析。结果表明,DGGE图谱中可检测到的8条带的16S rDNA基因序列中有7个基因序列与GenBank数据库登录的相关序列的相似性大于98%,余下的1个基因序列的相似性也大于96%。相似性大于98%的7个克隆中,有3个属于鞘氨醇杆菌属(Sphingobacterium),2个属于乳杆菌属(Lactobacillus),其它2个分别属于肠杆菌属(Enterobacter)和不动杆菌属(Acinetobacter)。首次报道了鞘氨醇杆菌作为优势菌群存在开菲尔Kefir粒中。     

应用rpoB和16S rDNA基因的变性梯度凝胶电泳技术对山羊瘤胃细菌多样性的研究

采用免培养的rpoB和16S rDNA基因的变性梯度凝胶电泳技术(DGGE)对3种山羊(波尔山羊,内蒙古绒山羊,四川南江黄羊)瘤胃细菌优势菌群结构进行了比较分析。研究结果显示rpoBDGGE图谱中条带数目少于16S rDNA图谱,并且条带分离效果明显,更有利于分析瘤胃细菌群落组成。从两种DGGE图谱中均可以发现3种山羊瘤胃细菌具有一定的相似性,种内个体间相似性明显高于种间相似性,这说明寄主品种是影响瘤胃细菌种群构成的一个重要因素。同时进行了部分优势细菌16S rDNA基因V6-V8区序列的系统发育分析。基因序列分析表明,DGGE图谱中优势条带的16S rDNA基因序列中有4条克隆的序列与基因库最相似菌的相似性大于97%,余下的克隆序列相似性在89%～96%之间,其中13条序列的与之相似性最高的序列均来自于未被鉴定的瘤胃细菌。     

变性梯度凝胶电泳法研究断奶仔猪粪样细菌区系变化

利用PCR和DGGE技术分析了12头仔猪在断奶后其粪样细菌区系的变化。粪样细菌16S rDNA的V6～V8可变区经PCR扩增,扩增产物经DGGE电泳后再进行相似性分析。结果表明,仔猪断奶当天粪样 DGGE谱带少,同窝仔猪间图谱相似。断奶后,随着断奶时间的推移,每头仔猪的DGGE图谱带逐渐增多,变得复杂和多样,仔猪个体间DGGE图谱差异逐渐增大。仔猪是否同窝以及所采食日粮类型对DGGE图谱没有明显影响。相似性分析还表明,日粮中添加寡果糖的仔猪在断奶后第1周,其粪样微生物区系变化迅速,而后缓慢。     

北极太平洋扇区海洋沉积物细菌多样性的系统发育分析

对北极太平洋扇区3个不同深度的海洋沉积物样品,采用PCR结合变性梯度凝胶电泳(DGGE)技术进行细菌16S rRNA基因V3区序列的系统发育分析。结果表明,同一个沉积物样品不同层次的DGGE电泳图谱不完全相同。从3个沉积物样品中共获得50条序列,大部分序列与从海洋环境尤其海洋沉积物获得的细菌16S rDNA序列相似性较高(88%~100%),归属于变形细菌(Proteobacteria)的gamma亚群、alpha亚群、beta亚群、epsilon亚群、delta亚群,Cytophaga_Flavobacterium_Bacteroides(CFB)群细菌和高G C含量的革兰氏阳性细菌等系统分类群,其中变形细菌(Proteobacteria)的gamma亚群为沉积物中的优势细菌类群。     

基于DGGE技术的茯砖茶发花过程细菌群变化分析

变性梯度胶电泳是当前微生物生态学研究重要技术之一。为研究茯砖茶发花过程中细菌群落结构和种类,对发花过程中不同时段细菌16S rDNA的V3可变区扩增,经变性梯度胶电泳(DGGE)后、对细菌DGGE条带进行克隆、测序和比对。结果表明,在发花过程的第0—4天、6—8天、10—14天茯砖茶发花存在3个差异较大的细菌优势种群结构的演变;从16SrDNA的V3可变区比对结果证明黑毛茶发花过程中有短波单胞菌属、诺卡氏菌属、新鞘脂菌属、突那梭菌属、韦龙氏假单胞菌属、乳杆菌属、克雷伯氏菌属以及不可培养ε-变形菌、腐败螺旋菌属、粘球菌属、根瘤菌属和6种未知分类地位的不可培养细菌,说明采用DGGE指纹图谱能更系统、更真实地反映茯砖茶发花发酵过程中细菌群落结构和多样性变化。     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

Biodiversity analysis of microbial community in the chem-bioflocculation treatment process

Total DNA was directly extracted from environmental samples and amplified with polymerase chain reaction (PCR) technique. The PCR products were fingerprinted via denaturing gradient gel electrophoresis (DGGE). Significant differences were observed in the microbial community structures between traditional treatment process and chem-bioflocculation process. The microbial community structure shift at different sampling locations in chem-bioflocculation process and on two typical operational conditions was studied. 16S rDNA V3 regions of some dominant species were sequenced and the species were identified. The microbial communities were stable in both the chem-bioflocculation process and the activated sludge process under various experimental conditions presented in this work. The attached growth treatment process was less stable when operational conditions changed.     

应用DGGE法对青海相邻两盐湖中细菌多样性的快速检测

分别对青海相邻两盐湖柯柯盐湖、茶卡盐湖土样、泥样进行富集培养后,从中提取的DNA用两套不同的细菌通用引物进行扩增,分别得到包含V8和V9高变区的16SrDNA片断。经变性梯度凝胶电泳(DGGE)分析,结果显示这两个盐湖富集样品中细菌多样性具有较大的差异,而且同一盐湖不同性质样品中的细菌多样性差异也较大,两湖的泥样富集样品中均表现出了稍丰富的多样性。     

新疆一号冰川土壤细菌多样性的研究

应用变性梯度凝胶电泳（DGGE）技术分离PCR扩增的16SrDNA来研究土壤微生物的多样性。直接从新疆一号冰川不同海拔高度的土壤样品中提取总DNA。用两套细菌通用引物分别扩增16SrDNA的V3和V6／V9高变区的特异性片段,PCR产物进行DGGE分析。PCR—DGGE图谱表明,PCR产物经DGGE检测到的条带清晰且分离效果好。结果表明,PCR—DGGE是一种快速研究微生物群落结构的有效方法。     

DG-DGGE分析产氢发酵系统微生物群落动态及种群多样性

应用双梯度-变性梯度凝胶电泳(DG-DGGE)对生物制氢反应器微生物种群的动态变化及多样性进行监测。间隔7d从反应器取厌氧活性污泥,以细菌16SrDNA通用引物进行V2～V3区域PCR扩增,长约450bp的PCR产物经DGGE分离后,获得污泥微生物群落的16SrDNA指纹图谱。污泥接种到反应器后微生物群落中既有原始种群的消亡和增长,也有次级种群的强化和演变。反应器在运行初期群落演替迅速,15d时微生物群落结构变化最大。群落结构的相似性随着演替时间的增加而逐渐升高,种群动态变化后形成稳定的群落结构。29d时微生物多样性基本保持不变,微生物优势种属达到19个OTU。在细菌竞争和协同作用制约下,种群多样性降低后趋于稳定,形成顶级群落。有些种群在群落结构中一直存在,是群落建成的原始种群,原始种群与次级种群在代谢过程中具有协同作用,表现出群落的综合生态特征。     

Molecular evaluation of microbial diversity occurring in different types of Mozzarella cheese

AIMS: The microbial community of different types of unripened Pasta Filata cheese was investigated by culture-independent methods with the aim of rapidly achieving knowledge about cheese microbiota and discriminating traditional and industrial cheeses. METHODS AND RESULTS: The microbial DNA extracted directly from the samples was used as a template in PCR experiments to amplify the 16S-23S rDNA spacer region and the V3 region of the 16S rDNA. Conventional electrophoresis of the amplified spacers allowed known classes of these DNA fragments belonging to genera and species of lactic acid bacteria to be distinguished. Denaturing gradient gel electrophoresis analysis of V3 amplicons was supported by reference cultures of LAB used as markers. CONCLUSION: Both molecular approaches furnished the expected information about microbial diversity and were quite valid for discriminating industrial, semi-artisanal or traditional cheeses, characterized by increasingly complex DNA profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods could be used for legal purposes when products obtained through prescribed manufacturing regulations are to be analysed.     

An outbreak of nonflocculating catabolic populations caused the breakdown of a phenol-digesting activated-sludge process.

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter-1 day-1 and then to 1.5 g liter-1 day-1. After the loading rate was increased to 1.5 g liter-1 day-1, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter-1 day-1. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the gyrB phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific gyrB sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter-1 day-1. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of gyrB-targeted fine population analyses in microbial ecology.     

应用变性梯度凝胶电泳和16SrDNA序列分析对kefir粒中细菌多样性的研究

以开菲尔（Kefir）粒为材料,经过DNA抽提和16SrDNA V3区PCR扩增,扩增产物经变性梯度凝胶电泳（DGGE）分离并切割电泳条带进行序列测定,并与现有的数据库进行了比较,对Kefir粒的细菌多样性进行分析。结果表明,DGGE图谱中可检测到的8条带的16SrDNA基因序列中有7个基因序列与GenBank数据库登录的相关序列的相似性大于98％,余下的1个基因序列的相似性也大于96％。相似性大于98％的7个克隆中,有3个属于鞘氨醇杆菌属（Sphingobacterium）,2个属于乳杆菌属（Lactobacillus）,其它2个分别属于肠杆菌属（Errterobacter）和不动杆菌属（Acinetobacter）。首次报道了鞘氨醇杆菌作为优势菌群存在开菲尔Kefir粒中。     

Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients.

A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils.