scholarly journals Distribution of Bifidobacterial Species in Human Intestinal Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific Primers

1999 ◽  
Vol 65 (10) ◽  
pp. 4506-4512 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Ryuichiro Tanaka ◽  
Masafumi Fukuda ◽  
Hiroshi Oyaizu
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Intestinal Tract ◽  
Bifidobacterium Longum ◽  
Rrna Gene ◽  
Human Feces ◽  
Specific Primers ◽  
Specific Pcr ◽  
Species Specific ◽  
Human Intestinal Tract

ABSTRACT In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis,B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum andB. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113–121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacteriumstrains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum andB. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, andB. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.

scholarly journals Development of 16S rRNA-Gene-Targeted Group-Specific Primers for the Detection and Identification of Predominant Bacteria in Human Feces

2002 ◽  
Vol 68 (11) ◽  
pp. 5445-5451 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Junji Fujimoto ◽  
Yukiko Miyamoto ◽  
Toshihiko Takada ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Intestinal Microflora ◽  
Quantitative Detection ◽  
Rrna Gene ◽  
Human Feces ◽  
Specific Primers ◽  
Specific Pcr ◽  
Detection And Identification ◽  
Bacterial Groups

ABSTRACT For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol. 68:2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.

Rapid identification of Saccharomonospora strains by multiplex PCR using species-specific primers within the 16S rRNA gene

1996 ◽  
Vol 27 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Jung-Hoon Yoon ◽  
Sung Taik Lee ◽  
Yong Kook Shin ◽  
Sam-Bong Kim ◽  
Hong-Joong Kim ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Multiplex Pcr ◽  
Rapid Identification ◽  
Rrna Gene ◽  
Specific Primers ◽  
Species Specific ◽  
The 16S Rrna Gene

Identification of non-tuberculous mycobacteria: utility of the GenoType Mycobacterium CM/AS assay compared with HPLC and 16S rRNA gene sequencing

2009 ◽  
Vol 58 (7) ◽  
pp. 900-904 ◽  
Author(s):  
Andie S. Lee ◽  
Peter Jelfs ◽  
Vitali Sintchenko ◽  
Gwendolyn L. Gilbert
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Gene Sequencing ◽  
Rapid Identification ◽  
Mycobacterium Abscessus ◽  
Clinical Isolates ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Clinically Significant ◽  
Species Specific

Non-tuberculous mycobacteria (NTM) causing clinical disease have become increasingly common and more diverse. A new reverse line probe assay, GenoType Mycobacterium CM/AS (Hain Lifescience), was evaluated for identification of a broad range of NTM. It was compared with phenotypic (HPLC) and molecular (DNA probes, in-house real-time multiplex species-specific PCR, 16S rRNA gene PCR and sequencing) identification techniques, which together provided the reference ‘gold standard’. A total of 131 clinical isolates belonging to 31 Mycobacterium species and 19 controls, including 5 non-Mycobacterium species, was used. Concordant results between the GenoType Mycobacterium assay and the reference identification were obtained in 119/131 clinical isolates (90.8 %). Identification of Mycobacterium abscessus and Mycobacterium lentiflavum by the assay was problematic. The GenoType Mycobacterium assay enables rapid identification of a broad range of potentially clinically significant Mycobacterium species, but some species require further testing to differentiate or confirm ambiguous results.

scholarly journals The development of a 16S rRNA gene based PCR for the identification of Streptococcus pneumoniaeand comparison with four other species specific PCR assays

2010 ◽  
Vol 10 (1) ◽  
Author(s):  
Nabil Abdullah El Aila ◽  
Stefan Emler ◽  
Tarja Kaijalainen ◽  
Thierry De Baere ◽  
Bart Saerens ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Pcr Assays ◽  
Species Specific

scholarly journals Analysis of the 16S rRNA Gene Sequence of Anaplasma centrale and Its Phylogenetic Relatedness to Other Ehrlichiae

2001 ◽  
Vol 8 (2) ◽  
pp. 241-244 ◽  
Author(s):  
Hisashi Inokuma ◽  
Yutaka Terada ◽  
Tugihiko Kamio ◽  
Didier Raoult ◽  
Philippe Brouqui
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Distance Analysis ◽  
Specific Pcr ◽  
Species Specific ◽  
Anaplasma Centrale ◽  
The 16S Rrna Gene

ABSTRACT The nucleotide sequence of the Anaplasma centrale 16S rRNA gene was determined and compared with the sequences of ehrlichial bacteria. The sequence of A. centrale was closely related to Anaplasma marginale by both level-of-similarity (98.08% identical) and distance analysis. A species-specific PCR was developed based upon the alignment data. The PCR can detect A. centrale DNA extracted from 10 infected bovine red blood cells in a reaction mixture. A. centrale DNA was amplified in the reaction, but not other related ehrlichial species.

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

2004 ◽  
Vol 70 (12) ◽  
pp. 7220-7228 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Junji Fujimoto ◽  
Toshihiko Takada ◽  
Ryuichiro Tanaka
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Intestinal Flora ◽  
Healthy Adults ◽  
Rrna Gene ◽  
Human Feces ◽  
Specific Primers ◽  
Wet Weight

ABSTRACT 16S rRNA gene-targeted group-specific primers were designed and validated for specific detection and quantification of the Clostridium leptum subgroup and the Atopobium cluster. To monitor the predominant bacteria in human feces by real-time PCR, we used these specific primers together with four sets of group-specific primers for the Clostridium coccoides group, the Bacteroides fragilis group, Bifidobacterium, and Prevotella developed in a previous study (T. Matsuki, K. Watanabe, J. Fujimoto, Y. Miyamoto, T. Takada, K. Matsumoto, H. Oyaizu, and R. Tanaka, Appl. Environ. Microbiol. 68:5445-5451, 2002). Examination of DNA extracted from the feces of 46 healthy adults showed that the C. coccoides group was present in the greatest numbers (log10 10.3 ± 0.3 cells per g [wet weight] [average ± standard deviation]), followed by the C. leptum subgroup (log10 9.9 ± 0.7 cells per g [wet weight]), the B. fragilis group (log10 9.9 ± 0.3 cells per g [wet weight]), Bifidobacterium (log10 9.4 ± 0.7 cells per g [wet weight]), and the Atopobium cluster (log10 9.3 ± 0.7 cells per g [wet weight]). These five bacterial groups were detected in all 46 volunteers. Prevotella was found in only 46% of the subjects at a level of log10 9.7 ± 0.8 cells per g (wet weight). Examination of changes in the population and the composition of the intestinal flora for six healthy adults over an 8-month period revealed that the composition of the flora of each volunteer remained stable throughout the test period.

scholarly journals Phylogenetic analysis of Helicobacter species based on partial gyrB gene sequences

2007 ◽  
Vol 57 (3) ◽  
pp. 444-449 ◽  
Author(s):  
Minna Hannula ◽  
Marja-Liisa Hänninen
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Primers ◽  
Phylogenetic Study ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Specific Pcr ◽  
Species Specific ◽  
The 16S Rrna Gene

Analysis of 16S rRNA gene sequences is one of the most common methods for investigating the phylogeny and taxonomy of bacteria. However, several studies have indicated that the 16S rRNA gene does not distinguish between certain Helicobacter species. We therefore selected for phylogenetic analysis an alternative marker, gyrB, encoding gyrase subunit B. The aim of this investigation was to examine the applicability of gyrB gene fragments (~1100 bp) for the phylogenetic study of 16 Helicobacter species and a total of 33 Helicobacter strains included in this study. Based on the sequenced fragments, a phylogenetic tree was obtained that contained two distinct clusters, with gastric species forming one cluster and enterohepatic species the other. The only exception was the gastric species Helicobacter mustelae, which clustered with the enterohepatic species. The calculated similarity matrix revealed the highest interspecies similarity between Helicobacter salomonis and Helicobacter felis (89 %) and the lowest similarity between Helicobacter pullorum and H. felis (60 %). The DNA G+C content of the sequenced fragments was ⩽40 mol% in enterohepatic species and >46 mol% in gastric species, excluding Helicobacter pylori and H. mustelae, with G+C contents of 34 and 42 mol%, respectively. In summary, the gyrB gene fragments provided superior resolution and reliability to the 16S rRNA gene for differentiating between closely related Helicobacter species. A further outcome of this study was achieved by designing gyrB gene-based species-specific PCR primers for the identification of Helicobacter bizzozeronii.

scholarly journals Atypical Helicobacter canadensis Strains Associated with Swine

2006 ◽  
Vol 72 (6) ◽  
pp. 4464-4471 ◽  
Author(s):  
G. Douglas Inglis ◽  
Malcolm McConville ◽  
Anno de Jong
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fragment Length Polymorphism ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Cardinal Characteristic ◽  
Swine Feces ◽  
Species Specific ◽  
Genomic Typing

ABSTRACT Forty-two Helicobacter isolates were isolated from swine feces in The Netherlands and Denmark. All 12 isolates sequenced (16S rRNA gene) formed a robust clade with Helicobacter canadensis (∼99% similarity). Species-specific PCR indicated that all of the isolates were H. canadensis isolates. Although the appearance of the porcine isolates was similar to the appearance of H. canadensis, only one of these isolates was able to hydrolyze indoxyl acetate, a cardinal characteristic of this taxon. Examination of the 23S rRNA and hsp60 genes revealed high levels of similarity between the porcine isolates and H. canadensis. However, amplified fragment length polymorphism genomic typing showed that isolates recovered from swine feces were genetically distinct from H. canadensis strains obtained from humans and geese.

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