Determination of fruit origin by using 26S rDNA fingerprinting of yeast communities by PCR–DGGE: preliminary application to Physalis fruits from Egypt

The determination of geographical origin is a demand of the traceability system of import–export food products. One hypothesis for tracing the source of a product is by global analysis of the microbial communities of the food and statistical linkage of this analysis to the geographical origin of the food. For this purpose, a molecular technique employing 26S rDNA profiles generated by PCR–DGGE was used to detect the variation in yeast community structures of three species of Physalis fruit (Physalis ixocarpa Brat, Physalis pubescens L, Physalis pruinosa L) from four Egyptian regions (Qalyoubia, Minufiya, Beheira and Alexandria Governments). When the 26S rDNA profiles were analysed by multivariate analysis, distinct microbial communities were detected. The band profiles of Physalis yeasts from different Governments were specific for each location and could be used as a bar code to discriminate the origin of the fruits. This method is a new traceability tool which provides fruit products with a unique biological bar code and makes it possible to trace back the fruits to their original location. Copyright © 2009 John Wiley & Sons, Ltd.     

提取鼠肠道内微生物基因组DNA的方法研究

通过改进鼠肠道微生物基因组DNA提取方法以期更全面地反映鼠肠道茵群的真实情况.采用CTAB和SDS结合的方法裂解细胞,同时改进了传统的酚/氯仿抽提方法,提取全过程控制在2 h以内.通过紫外分光光度计,细菌通用引物PCR,总菌群实时定量PCR,变性梯度凝胶电泳(DGGE)对改进方法及两种试剂盒法提取DNA的结果进行比较,评价了所建立的高效提取方法.与试剂盒相比,改进的方法DNA得率较高,简便快速,成本低廉.同时后续的PCR鉴定及DGGE菌群多样性分析显示,改进的方法可以更好地揭示鼠肠道菌群分布和特性.     

The impacts of cypermethrin pesticide application on the non-target microbial community of the pepper plant phyllosphere

Although pesticides have been extensively used for controlling insects and disease pathogens of plants, little is known regarding the impacts of applying these pesticides on the microbial community in the plant phyllosphere. Here, we report the effects of cypermethrin pesticide application upon the microbial community of the pepper plant phyllosphere. Assessments were made using culture-independent techniques including phospholipid fatty acid analysis (PLFA) and 16S rRNA gene directed Polymerase Chain Reaction with Denaturing Gradient Gel Electrophoresis (PCR-DGGE). During the 21 day greenhouse study, PLFA results indicated that both total and bacterial biomass increased after application of the pesticide. PLFA profiles also indicated that Gram-negative bacteria became predominant. DGGE analysis confirmed a significant change in bacterial community structure within the phyllosphere following the pesticide application where different dendrogram clusters were observed between control and treated samples. Phylogenetic analysis also suggested a change in bacterial phyla following treatment, where bands sequenced within control cultures were predominantly of the Firmicutes phylum, but those bands sequenced in the treated samples were predominantly members of the Bacteroidetes and γ-Proteobacteria phyla. In conclusion, this study revealed an increase in bacterial abundance and a shift in community composition within the pepper plant phyllosphere following the pesticide application, and highlighted the effective use of PLFA and PCR-DGGE for studying the effect of pesticides upon indigenous phyllosphere microbes.     

窖泥细菌群落结构演替及其与环境因子的相关性

利用PCR-DGGE系统研究了剑南春不同窖龄(2～50年)窖泥的微生物群落结构、种群演替趋势及其与环境因子的相关性。结果表明,2年窖龄窖泥的细菌丰度和多样性指数与5年样品相似,而10年窖龄窖泥的细菌丰度和多样性指数则明显增加,之后的10年基本持平,但50年时又显著下降。相同窖龄的窖泥样品中,中层的物种丰度和多样性指数均高于上层和下层,特别是在2～5年样品中这一趋势更加明显。DGGE和序列分析显示,窖泥中优势种群均分布在厚壁菌门(Phylum Firmicutes)的杆菌纲(Class Bacill)和梭菌纲(Class Clostrida),其中耐酸乳杆菌(Lactobacillus acetotolerans)、芽孢菌(Bacillus fordii)、未培养的瘤胃菌(uncultured Ruminococcaceae bacterium)和梭菌(uncultured Clostridia bacterium)为窖泥样品中的主要优势种群。典型对应分析显示,窖泥微生物群落结构与环境因子具有显著的相关性,其中有效磷、氨氮、pH对微生物群落结构的影响较大,其次是腐殖质,而水分含量的影响较小。     

基于DGGE分析的大鼠粪便及肠道细菌DNA提取方法研究

为获得高质量肠道细菌总DNA用于研究膳食纤维对大鼠肠道菌群的影响,本实验以SD大鼠为实验动物,灌胃番茄皮膳食纤维,收集大鼠粪便及盲肠,-70℃冰箱保存。分别采用Tiangen细菌基因组试剂盒法、蛋白酶K-十六烷基三甲基溴化铵(CTAB)法和溶菌酶法提取粪便和肠道中的细菌总DNA,通过琼脂糖凝胶电泳、细菌通用引物PCR扩增和变性梯度凝胶电泳(DGGE)对提取效果进行观察比较,发现将溶菌酶法进行改进后,可以获得满意效果。改进后的溶菌酶法与另外两种方法比较,具有成本低、时间短、DNA得率高和多样性好等特点;该方法获得的DNA样品适合于用DGGE技术分析大鼠粪便及肠道菌群。     

对虾养殖水体氨氧化菌的多样性分析与硝化作用的特征

为了研究珠三角地区不同空间分布的对虾养殖水体中氨氧化菌多样性的异同与探究养殖水体中氨氧化菌的硝化作用特征,应用PCR-DGGE技术,结合非加权算术平均法(UPGMA)聚类分析和多维尺度分析(MDS)的方法,对珠三角地区7个对虾养殖场的水样中氨氧化菌的多样性进行对比分析,继而构建了模拟养殖生态系统,评估了系统中微生物的硝化能力和硝化作用特征。结果表明:在养殖水体中,氨氧化古菌的类别明显少于氨氧化细菌,各样品间相似性较低,为41%到80%,没有高相似性的聚类;模拟系统中存在连续完整的亚硝化与硝化作用。     

降解水源水中有机物的优势菌筛选及其特性

为解决活性炭生物增强技术（BEAC）应用过程中接种高活性菌群稳定性差的问题,必须分离出既具有有机物降解能力,又具有种群竞争能力的菌株.应用变性梯度聚丙烯酰胺凝胶电泳（DGGE）技术,分析BAC滤池优势菌群结构,结果表明,有4种细菌占优势地位,其中2种可降解有机污染物,分别为Pseudomonas sp.和Bacillu...     

反硝化聚磷菌菌种筛分与除磷特性分析

目的 研究反硝化聚磷菌除磷特性,筛分同时具有反硝化和吸磷能力的菌种.方法 采用平板分离和DGGE技术相结合,对实验室反硝化除磷系统菌种进行分纯和菌种鉴定,并对富集培养后的每株单菌分别进行反硝化吸磷效果试验.结果 最终共得到15株单菌.结果 表明有ll株菌均具有不同程度反硝化吸磷能力,其中假单胞菌属的JB2和产碱菌属的JB3脱氮除磷效果最好,8 h后磷的去除量分别为13.76 mg/L和11.85 mg/L.DGGE试验结果表明,系统中反硝化聚磷菌优势种群可主要分为7个群,分别是Anaerolineae,Actinobacteria,Bacte-midetes,TM7,α-proteobactenia,δ-proteobacteria和γ-proteobacteria菌群.结论 Actinobacte-ria中的LB9号菌和γ-proteobacteria中的JB2号菌为反硝化除磷系统中占优势的反硝化聚磷菌.试验结果同时证明同一菌种可以同步完成反硝化和吸磷任务.     

水源受污染程度与微生物安全性调查研究

利用基因扩增-梯度变性凝胶电泳（PCR-DGGE）分子生物学方法对污染程度较轻的XL水库、污染程度中等的LZ湖和污染严重的XZ河等三类水源的微生物群落结构以及微生物安全性进行了调查研究.结果表明,PCR-DGGE方法能够检测出水中的微生物群落,便于准确评价水源的微生物安全性.16S rDNA鉴定表明,水源污染越严重,微生物群落结构越复杂、微生物含量越高、致病菌越多、微生物安全性越低.污染最严重的XZ河存在拟杆菌、假单胞菌、气单胞菌、苍白杆菌和黄色单胞菌等致病菌,LZ湖存在苍白杆菌和黄色单胞菌,XL水库存在可能产生藻毒素的微囊藻.保护水源及防治污染对于提高水源的微生物安全性具有关键作用.     

永川毛霉型豆豉在发酵过程中微生物总量与区系变化规律

采用巢式聚合酶链式反应配合变性梯度凝胶电泳技术对永川豆豉发酵过程中微生物区系生态演化进行解析。结果表明：永川豆豉在制曲过程中霉菌和细菌呈对数增长,进入后发酵阶段菌落总数快速下降,并保持在较低水平。永川豆豉在制曲前期有多种乳酸菌生长,后期乳酸菌受霉菌增长抑制,种类减少。在后发酵阶段奥德赛芽孢杆菌（Bacillus odysseyi）、乳酸杆菌（Lactobacillus oligofermentans）和乳酸杆菌（Lactobacillus lindneri）是优势菌群。同时在制曲初期也发现了费格森埃希菌（Escherichia fergusonii）等杂菌生长。永川豆豉制曲阶段优势霉菌是总状毛霉（Mucor racemosus）,同时伴有有性根霉（Rhizopus sexualis）、匍枝根霉（Rhizopus stolonifer）、大孢联轭霉（Syzygites megalocarpus）、米根霉（Rhizopus oryzae）的生长,后发酵阶段有接合酵母（Zygosaccharomyces sp.）的参与。