New real-time quantitative PCR procedure for quantification of bifidobacteria in human fecal samples - PubMed (original) (raw)

New real-time quantitative PCR procedure for quantification of bifidobacteria in human fecal samples

Miguel Gueimonde et al. Appl Environ Microbiol. 2004 Jul.

Abstract

The application of a real-time quantitative PCR method (5' nuclease assay), based on the use of a probe labeled at its 5' end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 x 10(4) cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces.

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Figures

FIG. 1.

FIG. 1.

Calibration curve obtained by plotting C t values as a linear function of the base 10 logarithm of the initial number of bifidobacteria (B. infantis DSM 20088T, B. longum JCM 1217T, and B. adolescentis JCM 1275T) in the culture determined by plate counting.

FIG. 2.

FIG. 2.

Results of the analysis of germfree rat feces spiked with known amounts of B. infantis DSM 20088. □, cells added; ○, cells detected by FISH; ×, cells detected by real-time PCR.

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References

    1. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. - PMC - PubMed
    1. Apajalahti, J. H., A. Kettunen, P. H. Nurminen, H. Jatila, and W. E. Holben. 2003. Selective plating underestimates abundance and shows differential recovery of bifidobacteria species from human feces. Appl. Environ. Microbiol. 69:5731-5735. - PMC - PubMed
    1. Benno, Y., and T. Mitsuoka. 1986. Development of intestinal microflora in humans and animals. Bifidobateria Microflora 5:13-25.
    1. Cole, J. R., B. Chai, T. L. Marsh, R. J. Farris, Q. Wang, S. A. Kulam, S. Chandra, D. M. McGarrell, T. M. Schmidt, G. M. Garrity, and J. M. Tiedje. 2003. The Ribosomal Database Project (RDP-II): previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res. 31:442-443. - PMC - PubMed
    1. Falk, P. G., L. V. Hooper, T. Midvedt, and J. I. Gordon. 1998. Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology. Microbiol. Mol. Biol. Rev. 62:1157-1170. - PMC - PubMed

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