Terminal Restriction Pattern Analysis of 16S rRNA Genes for the Characterization of Bacterial Communities of Activated Sludge.

Hiraishi, Akira; Iwasaki, Mitsuru; Shinjo, Hisashi

doi:10.1263/jbb.90.148

Cited by 36 publications

(37 citation statements)

References 36 publications

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“…The methods developed for numerical analysis of quinone profiles (Hiraishi et al, 1991;Iwasaki & Hiraishi, 1998) have been applied for processing T-RFLP data (Hiraishi et al, 2000). Differences in T-RFLP patterns among samples were evaluated using the dissimilarity (D) index (Hiraishi et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

“…This technique has been used for assessing the diversity and structure of complex bacterial communities in various environments (Clement et al, 1998;Dunbar et al, 2000; Gonzàlez et al, 2000;Hiraishi et al, 2000;Kaplan et al, 2001;Leser et al, 2000;Lukow et al, 2000;Moeseneder et al, 1999) and evaluated in separate review articles (Kitts, 2001;Marsh, 1999). In addition, the TAbbreviations: T-RF, terminal restriction fragment; T-RFLP, terminal restriction fragment length polymorphism.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis

Sakamoto

Takeuchi

Umeda

et al. 2003

Journal of Medical Microbiology

105

106

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Terminal restriction fragment-length polymorphism (T-RFLP) analysis was applied to characterize oral bacterial flora in saliva from 18 healthy subjects and 18 patients with periodontitis. The 16S rRNA genes (rDNAs) of oral bacteria and spirochaetes in saliva were amplified by PCR with a 69carboxy-fluorescein (6-FAM)-labelled universal forward primer (27F) and a universal reverse primer (1492R) or the Spirochaeta-selective reverse primer. The 16S rDNAs were digested with restriction enzymes with 4 bp recognition sites (HhaI or MspI) and analysed by using an automated DNA sequencer. T-RFLP patterns were numerically analysed using a computer program. From analysis of the oral bacterial community, patterns derived from periodontally healthy subjects and patients with periodontitis were grouped into different clusters, though with some uncertainty. Samples from patients with periodontitis tended to cluster into their respective types (aggressive and chronic periodontitis), although this was not very clear. Analysis of spirochaetal community using T-RFLP showed that the patterns derived from patients with periodontitis were grouped more as compared with the analysis of the oral bacterial community. These results suggest that samples from patients with periodontitis contain an unexpected diversity. T-RFLP patterns of 16S rDNAs from saliva samples of two periodontally healthy subjects over a 5-week period showed host-specific relatively stable oral bacterial flora. Our study indicates that T-RFLP analysis is useful for the assessment of diversity of oral bacterial flora and rapid comparison of the community structure between subjects with and without periodontitis. INTRODUCTIONA large number and variety of bacteria exist in the human oral cavity. However, analysis of these bacterial communities has been limited by conventional culture-dependent methods; thus, many oral bacteria remain uncultured and uncharacterized. Consequently, studies of causal microorganisms of oral diseases including periodontal disease are, in general, restricted to cultivable species.Recently, a phylogenetic approach based on 16S rRNA genes (rDNA) has been applied to investigate the diversity of cultivable and non-cultivable species in the human oral cavity without cultivation (Choi et al., 1994(Choi et al., , 1996Dymock et al., 1996;Kroes et al., 1999;Paster et al., 2001;Sakamoto et al., 2000;Spratt et al., 1999). The 16S rDNA clone library method can provide direct sequence information. Paster et al. (2001) demonstrated that the predominant subgingival microbial community consisted of 347 species or phylotypes, based on analysis of 2522 16S rRNA clones. However, analysis of individual 16S rDNA clones is an expensive and extremely inefficient approach for comparison of a multitude of bacterial communities.Terminal restriction fragment-length polymorphism (T-RFLP) is a new molecular approach that allows the assessment of a diversity of complex bacterial communities and rapid comparison of the community structure and diversity of different ecosyst...

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis

Sakamoto

Takeuchi

Umeda

et al. 2003

Journal of Medical Microbiology

105

106

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“…The discrepancies between the PCR data and the data of T-RFLP and DGGE analysis from samples B and S may have been caused by differences in the rRNA gene copy number. Hiraishi et al (7) demonstrated that the fluorescence intensity of T-RFs detected is proportional to the copy number of the 16S rRNA gene in each species rather than reflective of the relative content of different taxa. In addition, the discrepant data may be due to differences in the detection limits between the Bifidobacterium species-and group-specific primers and the Bifidobacterium genus-specific primers.…”

Section: Detection Of Bifidobacterial Species With Species-specific Pmentioning

confidence: 99%

Terminal Restriction Fragment Length Polymorphism Analysis for Human Fecal Microbiota and Its Application for Analysis of Complex Bifidobacterial Communities

Sakamoto

Hayashi

Benno

2003

Microbiology and Immunology

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A large number and variety of bacteria exist in the human intestinal tract. The balance of the intestinal microbiota closely relates to the health of the individual. Hence, understanding the human intestinal microbiota is very important. Traditional methods to determine the composition of the intestinal microbiota require various culture techniques, which are time-consuming and laborious. Therefore, many researchers used various culture-independent methods; including 16S rRNA probe hybridization (3, 23), PCR-related methods (29), and temperature gradient gel electrophoresis (TGGE) analysis (32), to characterize the human intestinal microbiota.A phylogenetic approach based on 16S rRNA (rDNA) has been applied to investigate the diversity of cultivable and non-cultivable species in human fecal samples without cultivation (4,24,30). The 16S rDNA clone library method can provide direct sequence information. We demonstrated previously that the predominant intestinal microbial community consisted of 130 species or "phylotypes"(17), which have been used for clusters of clone sequences that differed from known species by about 2% and were at least 99% similar to members of their cluster, based on analysis of 744 16S rRNA clones (4). However, analysis of individual 16S rDNA clones is an expensive and extremely inefficient approach for comparison of a multitude of bacterial communities.Terminal restriction fragment length polymorphism (T-RFLP) is a alternative molecular approach that allows the assessment of a diversity of complex bacterial communities and rapid comparison of the community structure and diversity of different ecosystems (11). We have already used this method to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis (20). In the field of intestinal microbiology, T-RFLP analysis is potentially useful for assessment of the diversity of the human fecal microbiota and rapid comparison of the community structure among individuals, but has not yet been reported to our knowledge.In the present study, we used T-RFLP analysis to characterize and compare human fecal microbiota among Abstract: Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to characterize and compare human fecal microbiota among individuals. T-RFLP patterns of fecal 16S ribosomal DNA (rDNA) PCR products from three adults revealed host-specific bacterial communities and were in good agreement with those reported in our previous study. In addition, we applied T-RFLP analysis for the analysis of complex bifidobacterial communities in human fecal samples. The developed method based on Bifidobacterium genus-specific PCR and T-RFLP could identify more than one bifidobacterial species. T-RFLP patterns of Bifidobacterium genus-specific PCR products from the fecal samples were host-specific as well as those of fecal 16S rDNA PCR products. These results were confirmed by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the genus Bifidobacterium and Bifidobacterium...

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“…One of the rRNA gene-based approaches is T-RFLP analysis, which allows the rapid identification of ribotypes from a variety of samples of environmental origin [16]. Due to the sensitivity and high throughput of this method, it is considered as an ideal technique for comparative community analyses [17].…”

Section: Introductionmentioning

confidence: 99%

Changes of catabolic genes and microbial community structures during biodegradation of nonylphenol ethoxylates and nonylphenol in natural water microcosms

Sei

Toyama

Ike

et al. 2008

Biochemical Engineering Journal

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Changes of possible key catabolic genes and microbial community structures during the degradation of NPEOs and NP in natural water microcosms were investigated using the most-probable-number-polymerase chain reaction (MPN-PCR) and terminal restriction fragment length polymorphism (T-RFLP). The copy number of catechol 2,3-dioxygenase (C23O) DNA increased significantly during NPEO and NP degradation, suggesting that meta-cleavage of the aromatic rings of NPEOs and NP might have happened. Catechol 1,2-dioxygenase (C12O) DNA, alkanecatabolic genes (alk), and 16S rDNA, on the other hand, did not change notably, suggesting that the two genes might not be the relevant genes for NPEOs and NP degradation. The 16S rRNA gene-based T-RFLP analysis results indicated that specific and different dominant (or degrading) bacteria should be selected, depending on the substances. A strain with a DNA length of 78 bp, which might be affiliated with the beta subclass of Proteobacteria, became the dominant species for NPEO degradation, while strains at 88 and 198 bp were dominant in the NP microcosm. Diversity of microbial community structure tended to be simplified after NPEO degradation, while that in the NP microcosm remained relatively stable. Five clusters were obtained according to the similarity in community structures of different microcosms by cluster analysis, which were consistent with the biodegradation behaviors of different microcosms. This is the first report on genetic evidence of a possible aromatic ring meta-cleaving pathway of NPEOs and NP in an aquatic environment.

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Terminal Restriction Pattern Analysis of 16S rRNA Genes for the Characterization of Bacterial Communities of Activated Sludge.

Cited by 36 publications

References 36 publications

Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis

Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis

Terminal Restriction Fragment Length Polymorphism Analysis for Human Fecal Microbiota and Its Application for Analysis of Complex Bifidobacterial Communities

Changes of catabolic genes and microbial community structures during biodegradation of nonylphenol ethoxylates and nonylphenol in natural water microcosms

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