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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| Abstract | 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. |
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| AbstractList | 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. 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. 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.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. |
| Author | Benno, Yoshimi Hayashi, Hidenori Sakamoto, Mitsuo |
| Author_xml | – sequence: 1 givenname: Hidenori surname: Hayashi fullname: Hayashi, Hidenori email: hayashi@jcm.riken.go.jp organization: Japan Collection of Microorganisms, RIKEN, 351-0198, Saitama, Japan – sequence: 2 givenname: Mitsuo surname: Sakamoto fullname: Sakamoto, Mitsuo organization: Japan Collection of Microorganisms, RIKEN, 351-0198, Saitama, Japan – sequence: 3 givenname: Yoshimi surname: Benno fullname: Benno, Yoshimi organization: Japan Collection of Microorganisms, RIKEN, 351-0198, Saitama, Japan |
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| References | Hold, G.L., Pryde, S.E., Russell, V.J., Furrie, E., and Flint, H.J. 2002. Assessment of microbial diversity in human colonic samples by 16S rDNA sequence analysis. FEMS Microbiol. Ecol. 39: 33-39. Hiraishi, A., Kamagata, Y., and Nakamura, K. 1995. Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA gene from methanogens. J. Ferment. Bioeng. 79: 523-529. Sghir, A., Gramet, G., Suau, A., Rochet, V., Pochart, P., and Dore, J. 2000. Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization. Appl. Environ. Microbiol. 66: 2263-2266. Langendijk, P.S., Schut, F., Jansen, G.J., Raangs, G.C., Kamphuis, G.R., Wilkinson, M.H., and Welling, G.W. 1995. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples. Appl. Environ. Microbiol. 61: 3069-3075. Moore, W.E., and Holdeman, L.V. 1974. Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl. Microbiol. 27: 961-979. Maidak, B.L., Cole, J.R., Parker, C.T. Jr., Garrity, G.M., Larsen, N., Li, B., Lilburn, T.G., McCaughey, M.J., Olsen, G.J., Overbeek, R., Pramanik, S., Schmidt, T.M., Tiedje, J.M., and Woese, C.R. 1999. A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res. 27: 171-173. Hayashi, H., Sakamoto, M., and Benno, Y. 2002. Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods. Microbiol. Immunol. 46: 535-548. Suau, A., Bonnet, R., Sutren, M., Godon, J.J., Gibson, G.R., Collins, M.D., and Dore, J. 1999. Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl. Environ. Microbiol. 65: 4799-4807. Nagashima, K., Hisada, T., Sato, M., and Mochizuki, J. 2003. Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces. Appl. Environ. Microbiol. 69: 1251-1262. Sakamoto, M., Hayashi, H., and Benno, Y. 2003. Terminal restriction fragment length polymorphism analysis for human fecal microbiota and its application for analysis of complex bifidobacterial communities. Microbiol. Immunol. 47: 133-142. Pearson, W.R., and Lipman, D.J. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. U.S.A. 85: 2444-2448. Wilson, K.H., and Blitchington, R.B. 1996. Human colonic biota studied by ribosomal DNA sequence analysis. Appl. Environ. Microbiol. 62: 2273-2278. Hayashi, H., Sakamoto, M., and Benno, Y. 2002. Fecal microbial diversity in a strict vegetarian as determined by molecular analysis and cultivation. Microbiol. Immunol. 46: 819-831. Hayashi, H., Sakamoto, M., Kitahara, M., and Benno, Y. 2003. Molecular analysis of fecal microbiota in elderly individuals using 16S rDNA library and T-RFLP. Microbiol. Immunol. 47: 557-570. Kitts, C.L. 2001. Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. Curr. Issues Intest. Microbiol. 2: 17-25. O'Sullivan, M.G., Thornton, G.C., O'Sullivan, G.C., and Collins, J.K. 1992. Probiotic bacteria myth or reality?. Trends Food Sci Technol. 31: 309-314. Mengoni, A., Grassi, E., and Bazzicalupo, M. 2002. Cloning method for taxonomic interpretation of T-RFLP patterns. BioTechniques 33: 990-991. Franks, A.H., Harmsen, H.J.M., Raangs, G.C., Jansen, G.J., Schut, F., and Welling, G.W. 1998. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 64: 3336-3345. 1974; 27 2002; 39 1995; 61 1995; 79 2002; 46 1999; 27 2000; 66 2002; 33 1998 2003; 47 2003; 69 1999; 65 1996; 62 2001; 2 1988; 85 1991 1992; 31 1998; 64 e_1_2_2_4_1 e_1_2_2_14_1 e_1_2_2_5_1 e_1_2_2_6_1 e_1_2_2_12_1 e_1_2_2_7_1 e_1_2_2_11_1 e_1_2_2_21_1 e_1_2_2_20_1 e_1_2_2_3_1 e_1_2_2_19_1 e_1_2_2_18_1 e_1_2_2_8_1 e_1_2_2_17_1 e_1_2_2_16_1 e_1_2_2_15_1 Ballongue J. (e_1_2_2_2_1) 1998 Lane D.J. (e_1_2_2_10_1) 1991 Kitts C.L. (e_1_2_2_9_1) 2001; 2 Mengoni A. (e_1_2_2_13_1) 2002; 33 |
| References_xml | – reference: O'Sullivan, M.G., Thornton, G.C., O'Sullivan, G.C., and Collins, J.K. 1992. Probiotic bacteria myth or reality?. Trends Food Sci Technol. 31: 309-314. – reference: Hayashi, H., Sakamoto, M., Kitahara, M., and Benno, Y. 2003. Molecular analysis of fecal microbiota in elderly individuals using 16S rDNA library and T-RFLP. Microbiol. Immunol. 47: 557-570. – reference: Sakamoto, M., Hayashi, H., and Benno, Y. 2003. Terminal restriction fragment length polymorphism analysis for human fecal microbiota and its application for analysis of complex bifidobacterial communities. Microbiol. Immunol. 47: 133-142. – reference: Kitts, C.L. 2001. Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. Curr. Issues Intest. Microbiol. 2: 17-25. – reference: Suau, A., Bonnet, R., Sutren, M., Godon, J.J., Gibson, G.R., Collins, M.D., and Dore, J. 1999. Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl. Environ. Microbiol. 65: 4799-4807. – reference: Hiraishi, A., Kamagata, Y., and Nakamura, K. 1995. Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA gene from methanogens. J. Ferment. Bioeng. 79: 523-529. – reference: Mengoni, A., Grassi, E., and Bazzicalupo, M. 2002. Cloning method for taxonomic interpretation of T-RFLP patterns. BioTechniques 33: 990-991. – reference: Hayashi, H., Sakamoto, M., and Benno, Y. 2002. Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods. Microbiol. Immunol. 46: 535-548. – reference: Maidak, B.L., Cole, J.R., Parker, C.T. Jr., Garrity, G.M., Larsen, N., Li, B., Lilburn, T.G., McCaughey, M.J., Olsen, G.J., Overbeek, R., Pramanik, S., Schmidt, T.M., Tiedje, J.M., and Woese, C.R. 1999. 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| Snippet | 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... 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... 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... |
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| SubjectTerms | 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 |
| Title | Evaluation of Three Different Forward Primers by Terminal Restriction Fragment Length Polymorphism Analysis for Determination of Fecal Bifidobacterium Spp. in Healthy Subjects |
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