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Applied and Environmental Microbiology, July 2004, p. 4165-4169, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4165-4169.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Miguel Gueimonde,1,2* Satu Tölkkö,1,2 Teemu Korpimäki,1 and Seppo Salminen1,2

Department of Biochemistry and Food Chemistry,1 Functional Foods Forum, University of Turku, Turku, Finland2

Received 12 November 2003/ Accepted 8 March 2004

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 104 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.

* Corresponding author. Mailing address: Department of Biochemistry and Food Chemistry, University of Turku, Vatselankatu 2, FIN-20014 Turku, Finland. Phone: 358 2 3336861. Fax: 358 2 3336884. E-mail: miguel.gueimonde{at}utu.fi.

Applied and Environmental Microbiology, July 2004, p. 4165-4169, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.4165-4169.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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