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1.
这是现代生物技术在环境微生物学中的应用系列综述文章的第三篇 ,讨论限制性片段长度多态性 (RFLP)分析、变性梯度凝胶电泳 (DGGE)和温度梯度凝胶电泳 (TGGE)以及报道基因。 相似文献
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Use of flow cytometric methods for single-cell analysis in environmental microbiology 总被引:3,自引:0,他引:3
Flow cytometry (FCM) is emerging as an important tool in environmental microbiology. Although flow cytometry applications have to date largely been restricted to certain specialized fields of microbiology, such as the bacterial cell cycle and marine phytoplankton communities, technical advances in instrumentation and methodology are leading to its increased popularity and extending its range of applications. Here we will focus on a number of recent flow cytometry developments important for addressing questions in environmental microbiology. These include (i) the study of microbial physiology under environmentally relevant conditions, (ii) new methods to identify active microbial populations and to isolate previously uncultured microorganisms, and (iii) the development of high-throughput autofluorescence bioreporter assays. 相似文献
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We have developed restriction fragment melting curve analyses (RFMCA), which is a novel method for the real-time analysis of microbial communities. The major advantage of RFMCA compared to, for example, terminal restriction fragment length polymorphism (T-RFLP) or temperature/denaturing gradient gel electrophoresis (TGGE/DGGE) is that the physical separation of DNA fragments is avoided. The RFMCA detection is done by melting point analyses in closed tube systems, which enables high-throughput applications. The robustness of RFMCA was demonstrated by analyzing both mixtures of known samples and the microbial communities in the cecal content of poultry. Our conclusions are that RFMCA is robust, gives a relatively high resolution, and has the potential for high-throughput explorative screenings of microbial communities and large clone libraries. 相似文献
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DNA restriction fragment polymorphism technologies such as amplified ribosomal DNA restriction analysis (ARDRA) and terminal
restriction fragment length polymorphism (T-RFLP) have been widely used in investigating microbial community structures. However,
these methods are limited due to either the low resolution or sensitivity. In this study, a fluorophore-ribosomal DNA restriction
typing (f-DRT) approach is developed for structural profiling of microbial communities. 16S rRNA genes are amplified from
the community DNA and digested by a single restriction enzyme Msp I. All restriction fragments are end-labeled with a fluorescent nucleotide Cy5-dCTP via a one-step extension reaction and
detected with an automated DNA sequencer. All 50 predicted restriction fragments between 100 and 600 bp were detected when
twelve single 16S rRNA gene sequences were analyzed using f-DRT approach; 92% of these fragments were determined with accuracy
of ±2 bp. In the defined model communities containing five components with different ratios, relative abundance of each component
was correctly revealed by this method. The f-DRT analysis also showed structural shifts of intestinal microbiota in carcinogen-treated
rats during the formation of precancerous lesions in the colon, as sensitive as multiple digestion-based T-RFLP analysis.
This study provides a labor and cost-saving new method for monitoring structural shifts of microbial communities. 相似文献
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Comparison of molecular fingerprinting methods for analysis of soil microbial community structure 总被引:1,自引:0,他引:1
Denaturing gradient gel electrophoresis (DGGE), terminal-restriction fragment length polymorphism (T-RFLP) analysis, and automated
ribosomal intergenic spacer analysis (ARISA) have been widely used as molecular fingerprinting methods for analysis of microbial
communities. To find suitable methods, we compared the three fingerprinting methods by analyzing soil fungal communities in
four differing land-use types: bare ground, crop fields, grasslands, and forests. We also examined optimal primer pairs for
DGGE analysis by comparing single and mixed DNA samples of cultured fungal populations. Principal coordinate analysis (PCO),
nonmetric multidimensional scaling method (NMDS), and analysis of similarities (ANOSIM), which are major multivariate statistical
analyses for quantifying fingerprint patterns, were compared. All three fingerprinting methods yielded clear discrimination
of soil fungal communities among the four land-use types, irrespective of statistical methods. The advantages and disadvantages
of the three fingerprinting methods were discussed. 相似文献
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Terminal restriction fragment length polymorphism (TRFLP) is a method that has been frequently used to survey the microbial diversity of environmental samples and to monitor changes in microbial communities. T-RFLP is a highly sensitive and reproducible procedure that combines a PCR with a labeled primer, restriction digestion of the amplified DNA, and separation of the terminal restriction fragment (T-RF). The reliable identification of T-RF requires the information of nucleotide sequences as well as the size of T-RF. However, it is difficult to obtain the information of nucleotide sequences because the T-RFs are fragmented and lack a priming site of 3'-end for efficient cloning and sequence analysis. Here, we improved on the T-RFLP method in order to analyze the nucleotide sequences of the distinct TRFs. The first method is to selectively amplify the portion of T-RF ligated with specific oligonucleotide adapters. In the second method, the termini of T-RFs were tailed with deoxynucleotides using terminal deoxynucleotidyl transferase (TdT) and amplified by a second round of PCR. The major T-RFs generated from reference strains and from T-RFLP profiles of activated sludge samples were efficiently isolated and identified by using two modified T-RFLP methods. These methods are less time consuming and labor-intensive when compared with other methods. The T-RFLP method using TdT has the advantages of being a simple process and having no limit of restriction enzymes. Our results suggest that these methods could be useful tools for the taxonomic interpretation of T-RFs. 相似文献
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Schütte UM Abdo Z Bent SJ Shyu C Williams CJ Pierson JD Forney LJ 《Applied microbiology and biotechnology》2008,80(3):365-380
Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies. 相似文献
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Lisa E. Mayhew Dennis J. Geist Susan E. Childers Jacob D. Pierson 《Geomicrobiology journal》2013,30(7-8):615-625
Microbial communities at six fumarole fields on Sierra Negra and Alcedo volcanoes in the Galápagos Islands were examined to test how extreme geochemical conditions affect microbial biodiversity. The geologic substrates consist of basalt and rhyolite with varying amounts of alteration and sulfur precipitates. Collected samples of substrates varied in pH from 0–6, and substrate temperatures were within the mesophilic, thermophilic, and hyperthermophilic temperature ranges. Terminal restriction fragment length polymorphism (T-RFLP) analyses were done to assess the relationship of communities to each other as a function of geologic substrate, pH, and temperature. Comparative analyses of community diversity define two distinct clusters showing that the relationship between spatially separated microbial communities at the fumaroles is most influenced by the pH of the local environment. 相似文献
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Microbial communities in hot pepper (Capsicum annuum L.) cultivation fields under different cultivation methods were investigated by terminal restriction fragment length polymorphism
(T-RFLP) analysis. Rhizosphere soil and leaf samples were collected from control, conventional and nature-friendly cultivation
fields between May and July, 2009. Two Bacillus subtilis strains were applied to nature-friendly cultivation fields as biocontrol agents during the sampling period. Relative abundances
of bacteria and plant pathogenic fungi related T-RFs were also measured to monitor the effect of biocontrol agents on potential
plant pathogenic fungi. In the principal component analysis (PCA) based on T-RFLP profiles, the microbial communities from
rhizosphere soil samples in July, including bacteria and fungi, showed distinct difference between nature-friendly cultivation
fields and other cultivation fields. However, there was no correlation between cultivation methods and leaf microbial communities
at any sampling period. Changes in the abundance of bacteria related T-RF in the rhizosphere of nature-friendly cultivation
fields were observed clearly two months after application of biocontrol agent, while the abundance of plant pathogenic fungi
related T-RFs significantly decreased. 相似文献
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Community structure analyses are more sensitive to differences in soil bacterial communities than anonymous diversity indices 总被引:1,自引:0,他引:1
Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities. 相似文献
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Community Structure Analyses Are More Sensitive to Differences in Soil Bacterial Communities than Anonymous Diversity Indices 总被引:4,自引:1,他引:3 下载免费PDF全文
Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities. 相似文献
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T L Marsh 《Current opinion in microbiology》1999,2(3):323-327
Terminal restriction fragment length polymorphism is a recent molecular approach that can assess subtle genetic differences between strains as well as provide insight into the structure and function of microbial communities. The technique has both high sensitivity and throughput making it ideal for comparative analyses. 相似文献
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Andrew D. Opatkiewicz David A. Butterfield & John A. Baross 《FEMS microbiology ecology》2009,70(3):81-92
The microbial community structure of five geographically distinct hydrothermal vents located within the Axial Seamount caldera, Juan de Fuca Ridge, was examined over 6 years following the 1998 diking eruptive event. Terminal restriction fragment length polymorphism (TRFLP) and 16S rRNA gene sequence analyses were used to determine the bacterial and archaeal diversity, and the statistical software primer v6 was used to compare vent microbiology, temperature and fluid chemistry. Statistical analysis of vent fluid temperature and composition shows that there are significant differences between vents in any year, but that the fluid composition changes over time such that no vent maintains a chemical composition completely distinct from the others. In contrast, the subseafloor microbial communities associated with individual vents changed from year to year, but each location maintained a distinct community structure (based on TRFLP and 16S rRNA gene sequence analyses) that was significantly different from all other vents included in this study. Epsilonproteobacterial microdiversity is shown to be important in distinguishing vent communities, while archaeal microdiversity is less variable between sites. We propose that persistent venting at diffuse flow vents over time creates the potential to isolate and stabilize diverse microbial community structures between vents. 相似文献
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Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities. 相似文献
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A.C. Pinheiro M.F. MacedoV. Jurado C. Saiz-JimenezC. Viegas J. BrandãoL. Rosado 《International biodeterioration & biodegradation》2011,65(4):619-627
This project was developed to fully assess the indoor air quality in archives and libraries from a fungal flora point of view. It uses classical methodologies such as traditional culture media - for the viable fungi - and modern molecular biology protocols, especially relevant to assess the non-viable fraction of the biological contaminants. Denaturing high-performance liquid chromatography (DHPLC) has emerged as an alternative to denaturing gradient gel electrophoresis (DGGE) and has already been applied to the study of a few bacterial communities. We propose the application of DHPLC to the study of fungal colonization on paper-based archive materials. This technology allows for the identification of each component of a mixture of fungi based on their genetic variation. In a highly complex mixture of microbial DNA this method can be used simply to study the population dynamics, and it also allows for sample fraction collection, which can, in many cases, be immediately sequenced, circumventing the need for cloning. Some examples of the methodological application are shown. Also applied is fragment length analysis for the study of mixed Candida samples. Both of these methods can later be applied in various fields, such as clinical and sand sample analysis. So far, the environmental analyses have been extremely useful to determine potentially pathogenic/toxinogenic fungi such as Stachybotrys sp., Aspergillus niger, Aspergillus fumigatus, and Fusarium sp. This work will hopefully lead to more accurate evaluation of environmental conditions for both human health and the preservation of documents. 相似文献
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Beth Orcutt Brad Bailey Hubert Staudigel Bradley M. Tebo Katrina J. Edwards 《Environmental microbiology》2009,11(7):1728-1735
We present an interlaboratory comparison between full-length 16S rRNA gene sequence analysis and terminal restriction fragment length polymorphism (TRFLP) for microbial communities hosted on seafloor basaltic lavas, with the goal of evaluating how similarly these two different DNA-based methods used in two independent labs would estimate the microbial diversity of the same basalt samples. Two samples were selected for these analyses based on differences detected in the overall levels of microbial diversity between them. Richness estimators indicate that TRFLP analysis significantly underestimates the richness of the relatively high-diversity seafloor basalt microbial community: at least 50% of species from the high-diversity site are missed by TRFLP. However, both methods reveal similar dominant species from the samples, and they predict similar levels of relative diversity between the two samples. Importantly, these results suggest that DNA-extraction or PCR-related bias between the two laboratories is minimal. We conclude that TRFLP may be useful for relative comparisons of diversity between basalt samples, for identifying dominant species, and for estimating the richness and evenness of low-diversity, skewed populations of seafloor basalt microbial communities, but that TRFLP may miss a majority of species in relatively highly diverse samples. 相似文献
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Singh BK Nazaries L Munro S Anderson IC Campbell CD 《Applied and environmental microbiology》2006,72(11):7278-7285
A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically. 相似文献