Terminal restriction pattern analysis of 16S rRNA genes for the characterization of bacterial communities of activated sludge

Hiraishi, Akira; Iwasaki, Mitsuru; Shinjo, Hisashi

doi:10.1016/s1389-1723(00)80102-4

Cited by 74 publications

(23 citation statements)

References 36 publications

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“…Both types of primer, however, are complementary and, when used together, provided a comprehensive snapshot of the status of microbial communities within environmental samples. The results described here, illustrate the use of these primers for T-RFLP, to provide high resolution and sensitivity for the detection of OTUs from a complex community, notwithstanding the potential technical problems of the method [14,52], including potential PCR bias [53,54].…”

Section: Discussionmentioning

confidence: 78%

“…[12,13]). One of these rRNA gene-based approaches is terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes, which allows for the rapid identi¢cation of ribotypes from a variety of samples, including soils and sludges of environmental origin [14]. Due to the sensitivity and high throughput of this method it is an ideal technique for comparative community analyses [11].…”

Section: Introductionmentioning

confidence: 99%

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Microbial community structure and methanogenic activity during start-up of psychrophilic anaerobic digesters treating synthetic industrial wastewaters

Collins

Woods

McHugh

et al. 2003

FEMS Microbiology Ecology

117

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Culture-independent, molecular techniques were applied to the characterization of microbial communities of an anaerobic granular sludge obtained from a full-scale digester. Procedures were optimised for total DNA recovery and polymerase chain reaction (PCR) amplification of 16S rDNA using archaea- and eubacteria-specific oligonucleotide primers. Cloned PCR products were subsequently screened by amplified rDNA restriction analysis to identify operational taxonomic units (OTUs). Inserts from clones representing each OTU were sequenced and phylogenetic trees were prepared. In addition, the microbial communities were characterised using terminal restriction fragment length polymorphism (T-RFLP). The specific methanogenic activity of the biomass, against various substrates, was also ascertained. Two anaerobic bioreactors were seeded with granular and non-granular (i.e. crushed) aliquots of the characterised sludge, respectively, and used to investigate the treatment of a volatile fatty acid (VFA)-based synthetic wastewater, at a loading rate of 5 kg COD m(-3) day(-1) at low ambient temperatures (18 degrees C). DNA was isolated from sludge samples during the test period and shifts in archaeal and eubacterial population structures were elucidated. The start-up period was successful with methane yields and COD removal efficiencies of 60-75% and 65-85%, respectively. Specific methanogenic activities of reactor biomass, obtained at the conclusion of the trial, indicated the development of psychrotolerant biomass during the 90-day experiment. Furthermore, the efficacy of T-RFLP as a molecular tool for use in the surveyance of engineered ecosystems was confirmed.

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Section: Discussionmentioning

confidence: 78%

Section: Introductionmentioning

confidence: 99%

Microbial community structure and methanogenic activity during start-up of psychrophilic anaerobic digesters treating synthetic industrial wastewaters

Collins

Woods

McHugh

et al. 2003

FEMS Microbiology Ecology

117

View full text Add to dashboard Cite

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“…With more complex artificial bifidobacterial communities containing 4 species however, there were instances where only the 2–3 most abundant species were detectable even though all species were within an order of magnitude of each other in abundance (Table S2). This type of bias is not unprecedented in TRFLP, and may be related to the fact that the detection of any given species by TRFLP is dependent on its rank abundance [16,37,38]. Given the strong similarity between the rrn templates of different bifidobacterial species this problem may be caused by chimera formation [39,40].…”

Section: Resultsmentioning

confidence: 99%

Use of bifidobacterial specific terminal restriction fragment length polymorphisms to complement next generation sequence profiling of infant gut communities

et al. 2013

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Bifidobacteria are intestinal anaerobes often associated with gut health. Specific bifidobacterial species are particularly common in the gastrointestinal tract of breast-fed infants. Current short read next-generation sequencing approaches to profile fecal microbial ecologies do not discriminate bifidobacteria to the species level. Here we describe a low-cost terminal restriction fragment length polymorphism (TRFLP) procedure to distinguish between the common infant-associated bifidobacterial species. An empirical database of TRF sizes was created from both common reference strains and well-identified isolates from infant feces. Species-specific quantitative PCR validated bifidobacterial-specific TRFLP profiles from infant feces. These results indicate that bifidobacterial-specific TRFLP is a useful method to monitor intestinal bifidobacterial populations from infant fecal samples. When used alongside next generation sequencing methods that detect broader population levels at lower resolution, this high-throughput, low-cost tool can help clarify the role of bifidobacteria in health and disease.

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“…This technique relies on the inherent variation of the sequence of a molecular marker [30] and is the most widely used method in identifying phylogenetic specificity in bacterial communities [31]. T-RFLP analysis includes PCR amplification, using one primer that is fluorescently end-labelled, restriction enzyme digestion of the amplicon and detection of the terminal restriction fragment by an automated DNA sequencer or capillary electrophoresis [31].…”

Section: Molecular Tools For Community Studiesmentioning

confidence: 99%

“…T-RFLP analysis includes PCR amplification, using one primer that is fluorescently end-labelled, restriction enzyme digestion of the amplicon and detection of the terminal restriction fragment by an automated DNA sequencer or capillary electrophoresis [31]. The resulting output consists of a microbial profile where each detected length is that of specific fragments from the digested PCR product.…”

Section: Molecular Tools For Community Studiesmentioning

confidence: 99%

Approaches to analyse dynamic microbial communities such as those seen in cystic fibrosis lung

et al. 2009

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Microbial communities play vital roles in many aspects of our lives, although our understanding of microbial biogeography and community profiles remains unclear. The number of microbes or the diversity of the microbes, even in small environmental niches, is staggering. Current microbiological methods used to analyse these communities are limited, in that many microorganisms cannot be cultured. Even for the isolates that can be cultured, the expense of identifying them definitively is much too high to be practical. Many recent molecular technologies, combined with bioinformatic tools, are raising the bar by improving the sensitivity and reliability of microbial community analysis. These tools and techniques range from those that attempt to understand a microbial community from their length heterogeneity profiles to those that help to identify the strains and species of a random sampling of the microbes in a given sample. These technologies are reviewed here, using the microbial communities present in the lungs of cystic fibrosis patients as a paradigm.

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Terminal restriction pattern analysis of 16S rRNA genes for the characterization of bacterial communities of activated sludge

Cited by 74 publications

References 36 publications

Microbial community structure and methanogenic activity during start-up of psychrophilic anaerobic digesters treating synthetic industrial wastewaters

Microbial community structure and methanogenic activity during start-up of psychrophilic anaerobic digesters treating synthetic industrial wastewaters

Use of bifidobacterial specific terminal restriction fragment length polymorphisms to complement next generation sequence profiling of infant gut communities

Approaches to analyse dynamic microbial communities such as those seen in cystic fibrosis lung

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