New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

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 × 10

Applied and Environmental Microbiology.2004:70(7):4165

Department of Biochemistry and Food Chemistry,

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