Restriction fragment length polymorphism analysis ofFusobacterium necrophorumusing a novel repeat DNA sequence and a 16S rRNA gene probe

1993 ◽  
Vol 107 (2-3) ◽  
pp. 205-210 ◽  
Author(s):  
A.L.M. Hodgson ◽  
L.A. Nicholson ◽  
T.J. Doran ◽  
L.A. Corner
2003 ◽  
Vol 69 (2) ◽  
pp. 1251-1262 ◽  
Author(s):  
Koji Nagashima ◽  
Takayoshi Hisada ◽  
Maremi Sato ◽  
Jun Mochizuki

ABSTRACT New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.


2006 ◽  
Vol 72 (5) ◽  
pp. 3788-3792 ◽  
Author(s):  
Julie C. Frey ◽  
Jessica M. Rothman ◽  
Alice N. Pell ◽  
John Bosco Nizeyi ◽  
Michael R. Cranfield ◽  
...  

ABSTRACT We describe the bacterial diversity in fecal samples of a wild gorilla by use of a 16S rRNA gene clone library and terminal-restriction fragment length polymorphism (T-RFLP). Clones were classified as Firmicutes, Verrucomicrobia, Actinobacteria, Lentisphaerae, Bacteroidetes, Spirochetes, and Planctomycetes. Our data suggest that fecal populations did not change temporally, as determined by T-RFLP.


2005 ◽  
Vol 54 (11) ◽  
pp. 1093-1101 ◽  
Author(s):  
Hidenori Hayashi ◽  
Rei Takahashi ◽  
Takahiro Nishi ◽  
Mitsuo Sakamoto ◽  
Yoshimi Benno

Microbiota in gut contents of jejunum, ileum, caecum and recto-sigmoid colon obtained from three elderly individuals at autopsy were compared using 16S rRNA gene libraries and terminal restriction fragment length polymorphism (T-RFLP). Random clones of 16S rRNA gene sequences were isolated after PCR amplification with universal primer sets of total genomic DNA extracted from each sample of gut contents. An average of 90 randomly selected clones were partially sequenced (about 500 bp). T-RFLP analysis was performed using the 16S rRNA gene amplified from each sample. The lengths of the terminal restriction fragments were analysed after digestion with HhaI and MspI. The jejunal and ileal microbiota consisted of simple microbial communities of streptococci, lactobacilli, ‘Gammaproteobacteria', the Enterococcus group and the Bacteroides group. Most of the species were facultative anaerobes or aerobes. The Clostridium coccoides group and the Clostridium leptum subgroup, which are the most predominant groups in human faeces, were not detected in samples from the upper gastrointestinal tract. The caecal microbiota was more complex than the jejunal and ileal microbiota. The C. coccoides group, the C. leptum subgroup and the Bacteroides group were detected in the caecum. The recto-sigmoidal colonic microbiota consisted of complex microbial communities, with numerous species that belonged to the C. coccoides group, the C. leptum subgroup, the Bacteroides group, ‘Gammaproteobacteria', the Bifidobacterium group, streptococci and lactobacilli, and included more than 26 operational taxonomic units. The results showed marked individual differences in the composition of microbiota in each region.


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