Evaluation of Three Different Forward Primers by Terminal Restriction Fragment Length Polymorphism Analysis for Determination of Fecal Bifidobacterium Spp. in Healthy Subjects

The 27F forward primer is frequently used in 16S rRNA gene libraries and T‐RFLP analysis. However, Bifidobacterium spp. were barely detected with this primer in human fecal samples. In this study, fecal microbiota were analyzed using the T‐RFLP method with three different forward primers (27F, 35F,...

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Vydáno v:Microbiology and immunology Ročník 48; číslo 1; s. 1 - 6
Hlavní autoři: Hayashi, Hidenori, Sakamoto, Mitsuo, Benno, Yoshimi
Médium: Journal Article
Jazyk:angličtina
Vydáno: Australia Blackwell Publishing Ltd 01.01.2004
Témata:
Adult
Bifidobacterium
Bifidobacterium - classification
Bifidobacterium - genetics
Bifidobacterium - isolation & purification
DNA Primers
DNA, Bacterial - analysis
DNA, Bacterial - chemistry
DNA, Bacterial - isolation & purification
DNA, Ribosomal - analysis
DNA, Ribosomal - chemistry
DNA, Ribosomal - isolation & purification
Feces - microbiology
Female
forward primer
human fecal microbiota
Humans
Male
Middle Aged
Polymerase Chain Reaction
Polymorphism, Restriction Fragment Length
RNA, Ribosomal, 16S - genetics
Sequence Analysis, DNA
Species Specificity
spp
T-RFLP
ISSN:0385-5600, 1348-0421
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Shrnutí:The 27F forward primer is frequently used in 16S rRNA gene libraries and T‐RFLP analysis. However, Bifidobacterium spp. were barely detected with this primer in human fecal samples. In this study, fecal microbiota were analyzed using the T‐RFLP method with three different forward primers (27F, 35F, and 529F) in conjunction with one reverse primer (1492R). T‐RFLP analysis of fecal microbiota using 35F and 529F detected higher proportions of the terminal restriction fragment (T‐RF) corresponding to Bifidobacterium spp. than that using 27F. 27F is in imperfect agreement while 35F and 529F are in good concordance with the 16S rRNA gene sequences of Bifidobacterium spp., and the latter primers allowed for the detection of T‐RFs of Bifidobacterium spp. in fecal samples from five healthy subjects. The T‐RFs presumed to be Bifidobacterium spp. were cloned and sequenced, and found to match the 16S rRNA gene sequences of Bifidobacterium spp. Among the five fecal samples, two samples with low frequencies of T‐RFs of Bifidobacterium spp. were detected using these forward primers. This probably reflects a low prevalence of Bifidobacterium spp. in these two samples. Our study emphasizes the importance of selecting a suitable forward primer for detection of Bifidobacterium spp.
Bibliografie:ark:/67375/WNG-Z1L0GKBG-M
istex:B304B924CBAFE6247E453B52E5818EEFFC536DB0
Special Postdoctoral Research Program of RIKEN, Saitama, Japan
ArticleID:MIM03481
ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ObjectType-Article-2
ObjectType-Undefined-1
ObjectType-Feature-3
ISSN:0385-5600
1348-0421
DOI:10.1111/j.1348-0421.2004.tb03481.x