Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Human Feces

Matsuki, Takahiro; Watanabe, Koichi; Fujimoto, Junji; Takada, Toshihiko; Tanaka, Ryuichiro

doi:10.1128/aem.70.12.7220-7228.2004

Cited by 576 publications

(328 citation statements)

References 39 publications

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“…control subjects (P=0?05) (Table 3). Altogether, there was mostly smaller temporal variation in the studied clostridial groups than interindividual variation, and this has also been reported by Matsuki et al (2004). However, the instability of predominant bacteria was also seen in the change of the proportions of the studied clostridial groups (Tables 2 and Table 3.…”

Section: Temporal Stability Of Clostridial Populationsmentioning

confidence: 58%

Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria

Maukonen

Satokari

Mättö

et al. 2006

Journal of Medical Microbiology

132

125

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The differences in faecal bacterial population between irritable bowel syndrome (IBS) and control subjects have been reported in several studies. The aim of the present study was to compare the predominant and clostridial faecal microbiota of IBS subjects and healthy controls by applying denaturing gradient gel electrophoresis (DGGE) and a recently developed multiplexed and quantitative hybridization-based technique, transcript analysis with the aid of affinity capture (TRAC). According to the results, the studied clostridial groups (Clostridium histolyticum, Clostridium coccoides-Eubacterium rectale, Clostridium lituseburense and Clostridium leptum) represented the dominant faecal microbiota of most of the studied subjects, comprising altogether 29-87 % of the total bacteria as determined by the hybridized 16S rRNA. The C. coccoides-E. rectale group was the dominant subgroup of clostridia, contributing a mean of 43 % of the total bacteria in control subjects and 30 % (constipation type) to 50 % (diarrhoea type) in different IBS symptom category subjects. The proportion of the C. coccoides-E. rectale group was found to be significantly lower in the constipation-type IBS subjects than in the control subjects. DNA-based PCR-DGGE and RNA-based RT-PCR-DGGE analyses targeted to the predominant bacterial population showed considerable biodiversity as well as uniqueness of the microbiota in each subject, in both control and IBS subject groups. The RT-PCR-DGGE profiles of the IBS subjects further indicated higher instability of the bacterial population compared to the control subjects. The observations suggest that clostridial microbiota, in addition to the instability of the active predominant faecal bacterial population, may be involved in IBS. INTRODUCTIONThe composition of the resident intestinal microbiota varies between individuals, and the predominant population is fairly stable under normal conditions (Zoetendal et al., 1998; Harmsen et al., 2002a;Vanhoutte et al., 2004; Mättö et al., 2005). However, several factors, such as antibiotic therapy, ageing and disease, may cause disturbances in the intestinal balance. Transient disturbance of the intestinal microbiota during antibiotic therapy has been shown in several studies (Edlund & Nord, 2000; Donskey et al., 2003). Changes in the intestinal microbiota have also been suggested to occur in certain intestinal diseases and disorders, such as inflammatory bowel disease (IBD) (Seksik et al., 2003) and irritable bowel syndrome (IBS) (Madden & Hunter, 2002). IBS is an intestinal disorder that involves continuous or recurrent intestinal pain or discomfort that is relieved during defecation. In addition, IBS symptoms include bloating, altered stool frequency, form or passage, and passage of mucus (Thompson et al., 1999). The existence of abnormal colonic fermentation in IBS (King et al., 1998) and alleviation of IBS symptoms by eradication of small intestinal bacterial overgrowth by antibiotic therapy (Pimentel et al., 2000), suggest that the intestinal microbiota has...

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Section: Temporal Stability Of Clostridial Populationsmentioning

confidence: 58%

Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria

Maukonen

Satokari

Mättö

et al. 2006

Journal of Medical Microbiology

132

125

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“…The 'Atopobium cluster'-specific 16S rRNA gene-targeted primers of Matsuki et al (2004) were employed, but with a GC clamp attached to primer c-Atopo-F [GCc-Atopo-F, 59-CGCCCGCCGCGCGCGGCG-GGCGGGGCGGGGGCACGGGGGGGGGTTGAGAGACCGACC-39 (Hoyles, 2009); c-Atopo-R, 59-GGACGTCTTCTTCGRGGC-39]. Reaction mixtures (50 ml) contained 10 ml of 56 GoTaq Flexi Buffer (Promega), 5 ml of dNTPs (12.5 mM each; Promega), 2 ml of MgCl 2 (25 mM; Promega), 1 ml of each primer (20 pmol; Sigma Genosys), 1 ml of Taq polymerase (1.25 U; Promega), and 1 ml DNA.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, a single colony was suspended in 10 ml of filter-sterilized H 2 O using a sterile toothpick. The cell suspension was microwaved at high temperature for 30 s (Panasonic NN-T221MBBPQ) and used as DNA template for 'Atopobium cluster'-specific PCR using the primers of Matsuki et al (2004). Inhouse strains were used as negative (Megasphaera sp.…”

Section: Methodsmentioning

confidence: 99%

“…representation as determined by quantitative PCR (qPCR) and FISH (Harmsen et al, 2000(Harmsen et al, , 2002Matsuki et al, 2004). It is well known that Actinobacteria, specifically bifidobacteria, are under-represented or remain undetected in PCRbased studies, with factors such as selection of DNA extraction method, PCR primers and cycling conditions affecting their representation in clone libraries (Wilson & Blitchington, 1996;Suau et al, 1999;Koenig et al, 2011;Maukonen et al, 2012;Sim et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Dynamics and diversity of the ‘Atopobium cluster' in the human faecal microbiota, and phenotypic characterization of ‘Atopobium cluster' isolates

2015

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This study monitored the dynamics and diversity of the human faecal 'Atopobium cluster' over a 3-month period using a polyphasic approach. Fresh faecal samples were collected fortnightly from 13 healthy donors (six males and seven females) aged between 26 and 61 years. FISH was used to enumerate total (EUB338mix) and 'Atopobium cluster' (ATO291) bacteria, with counts ranging between 1.12¾10 11 and 9.95¾10 11 , and 1.03¾10 9 and 1.16¾10 11 cells (g dry weight faeces) "1 , respectively. The 'Atopobium cluster' population represented 0.2-22 % of the total bacteria, with proportions donor-dependent. Denaturing gradient gel electrophoresis (DGGE) using 'Atopobium cluster'-specific primers demonstrated faecal populations of these bacteria were relatively stable, with bands identified as Collinsella aerofaciens, Collinsella intestinalis/ Collinsella stercoris, Collinsella tanakaei, Coriobacteriaceae sp. PEAV3-3, Eggerthella lenta, Gordonibacter pamelaeae, Olsenella profusa, Olsenella uli and Paraeggerthella hongkongensis in the DGGE profiles of individuals. Colony PCR was used to identify 'Atopobium cluster' bacteria isolated from faeces (n5224 isolates). 16S rRNA gene sequence analysis of isolates demonstrated Collinsella aerofaciens represented the predominant (88 % of isolates) member of the 'Atopobium cluster' found in human faeces, being found in nine individuals. Eggerthella lenta was identified in three individuals (3.6 % of isolates). Isolates of Collinsella tanakaei, an 'Enorma' sp. and representatives of novel species belonging to the 'Atopobium cluster' were also identified in the study. Phenotypic characterization of the isolates demonstrated their highly saccharolytic nature and heterogeneous phenotypic profiles, and 97 % of the isolates displayed lipase activity.

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“…Real-time quantitative PCR can be used for more specific microbial DNA quantification. [58][59][60][61][62][63] A more comprehensive analysis is carried out using the 454 pyrosequencing 16S ribosomal RNA method, which is currently one of the best techniques for identification of microbes that cannot be analyzed or isolated by traditional culture-based methods. This technology is available and is currently being used to study and better understand the composition, function and effects of medical interventions in the intestinal microbiota.…”

Section: Microbial Components That Modulate Inflammationmentioning

confidence: 99%

The developing intestinal microbiome and its relationship to health and disease in the neonate

2011

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Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Human Feces

Cited by 576 publications

References 39 publications

Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria

Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria

Dynamics and diversity of the ‘Atopobium cluster' in the human faecal microbiota, and phenotypic characterization of ‘Atopobium cluster' isolates

The developing intestinal microbiome and its relationship to health and disease in the neonate

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