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J Microbiol Methods. 2004 Oct;59(1):53-69.

T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages.

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Laboratory of Food Hygiene, Department of Food Technology, Engineering and Nutrition, P.O. Box 124, Lund SE-22100, Sweden.


The fecal microbiota of two healthy Swedish infants was monitored over time by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Principal component analysis (PCA) of the T-RFLP profiles revealed that the fecal flora in both infants was quite stable during breast-feeding and a major change occurred after weaning. The two infants had different sets of microbiota at all sampling time points. 16S rDNA clone libraries were constructed and the predominant terminal restriction fragments (T-RFs) were identified by comparing T-RFLP patterns in the fecal community with that of corresponding 16S rDNA clones. Sequence analysis indicated that the infants were initially colonized mostly by members of Enterobacteriaceae, Veillonella, Enterococcus, Streptococcus, Staphylococcus and Bacteroides. The members of Enterobacteriaceae and Bacteroides were predominant during breast-feeding in both infants. However, Enterobacteriaceae decreased while members of clostridia increased after weaning. T-RFLP in combination with PCA and 16S rRNA gene sequencing was shown to be an effective strategy for comparing fecal microbiota in infants and pointing out the major changes.

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