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

2015 ◽  
Vol 81 (19) ◽  
pp. 6749-6756 ◽  
Author(s):  
Yun-Wen Yang ◽  
Mang-Kun Chen ◽  
Bing-Ya Yang ◽  
Xian-Jie Huang ◽  
Xue-Rui Zhang ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Chronic Inflammatory Bowel Disease ◽  
Trinitrobenzene Sulfonic Acid ◽  
Rrna Gene ◽  
Specific Primers ◽  
Content Type

ABSTRACTMouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers forFirmicutes,Actinobacteria,Bacteroidetes,Deferribacteres, “CandidatusSaccharibacteria,”Verrucomicrobia,Tenericutes, andProteobacteriawere designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level.

scholarly journals Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

2004 ◽  
Vol 70 (1) ◽  
pp. 167-173 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Junji Fujimoto ◽  
Yukiko Kado ◽  
Toshihiko Takada ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Intestinal Flora ◽  
Healthy Adults ◽  
Pcr Detection ◽  
Distribution Analysis ◽  
Specific Primers ◽  
Cell Counts ◽  
Species Specific

ABSTRACT A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 106 to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >106 cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

Real-time PCR of the 16S-rRNA gene in the diagnosis of neonatal bacteraemia

2008 ◽  
Vol 97 (10) ◽  
pp. 1376-1380 ◽  
Author(s):  
Andreas Ohlin ◽  
Anders Bäckman ◽  
Maria Björkqvist ◽  
Paula Mölling ◽  
Margaretha Jurstrand ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Rrna Gene ◽  
The 16S Rrna Gene

scholarly journals Prevalence of Swine Hemoplasmas Revealed by Real-Time PCR Using 16S rRNA Gene Primers

2012 ◽  
Vol 74 (10) ◽  
pp. 1315-1318 ◽  
Author(s):  
Yusaku WATANABE ◽  
Masatoshi FUJIHARA ◽  
Jin SUZUKI ◽  
Fumina SASAOKA ◽  
Kazuya NAGAI ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Rrna Gene

Impact of a probiotic, inulin, or their combination on the piglets’ microbiota at different intestinal locations

2015 ◽  
Vol 6 (4) ◽  
pp. 473-483 ◽  
Author(s):  
V.A. Sattler ◽  
K. Bayer ◽  
G. Schatzmayr ◽  
A.G. Haslberger ◽  
V. Klose
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Real Time ◽  
Relative Abundance ◽  
Real Time Pcr ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Feed Additives ◽  
Rrna Gene ◽  
16S Rrna Gene Pyrosequencing

Natural feed additives are used to maintain health and to promote performance of pigs without antibiotics. Effects of a probiotic, inulin, and their combination (synbiotic), on the microbial diversity and composition at different intestinal locations were analysed using denaturing gradient gel electrophoresis (DGGE), real-time PCR, and 16S rRNA gene pyrosequencing. Bacterial diversity assessed by DGGE and/or pyrosequencing was increased by inulin in all three gut locations and by the synbiotic in the caecum and colon. In contrast, the probiotic did only affect the microbiota diversity in the ileum. Shifts in the DGGE microbiota profiles of the caecum and colon were detected for the pro- and synbiotic fed animals, whereas inulin profiles were more similar to the ones of the control. 16S rRNA gene pyrosequencing revealed that all three additives could reduce Escherichia species in each gut location, indicating a potential beneficial effect on the gut microbiota. An increase of relative abundance of Clostridiaceae in the large intestine was found in the inulin group and of Enterococcaceae in the ileum of probiotic fed pigs. Furthermore, real-time PCR results showed that the probiotic and synbiotic increased bifidobacterial numbers in the ileum, which was supported by sequencing results. The probiotic and inulin, to different extents, changed the diversity, relative abundance of phylotypes, and community profiles of the porcine microbiota. However, alterations of the bacterial community were not uniformly between gut locations, demonstrating that functionality of feed additives is site specific. Therefore, gut sampling from various locations is crucial when investigations aim to identify the composition of a healthy gut microbiota after its manipulation through feed additives.

scholarly journals Haemotropic Mycoplasma Infection Revealed by Real-Time PCR in Specific Pathogen-Free Rats

2014 ◽  
Vol 58 (3) ◽  
pp. 375-378 ◽  
Author(s):  
Hinako Sashida ◽  
Ryô Harasawa ◽  
Toshihiro Ichijo ◽  
Hiroshi Satoh ◽  
Kazuhisa Furuhama
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Wistar Rat ◽  
Rrna Gene ◽  
Specific Pathogen Free ◽  
Sprague Dawley ◽  
Laboratory Rats ◽  
Specific Pathogen

Abstract The presence of Mycoplasma haemomuris (haemoplasma) in blood samples collected from specific pathogen-free (SPF) laboratory rats bred in Japan was reported. Its presence was examined in Fischer 344, Sprague-Dawley (SD), and Wistar rat strains of both sexes by real-time PCR. All strains were positive for M. haemomuris infection. The 16S rRNA gene of M. haemomuris strain detected in the animals was amplified using end-point PCR. Only the entire nucleotide sequence of 16S rRNA gene of a mycoplasma strain detected in SD rats was determined and compared to those of other haemoplasmas. Our investigations suggest a wide M. haemomuris infection among the SPF rats purchased from commercial breeders in Japan.

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