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Journal of Clinical Microbiology, June 2004, p. 2566-2572, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2566-2572.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora

Stephan J. Ott,1,{dagger} Meike Musfeldt,1,{dagger} Uwe Ullmann,2 Jochen Hampe,1 and Stefan Schreiber1*

Department of General Internal Medicine,1 Institute of Medical Microbiology and Virology, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany2

Received 18 February 2003/ Returned for modification 5 September 2003/ Accepted 18 February 2004

The composition of the human intestinal flora is important for the health status of the host. The global composition and the presence of specific pathogens are relevant to the effects of the flora. Therefore, accurate quantification of all major bacterial populations of the enteric flora is needed. A TaqMan real-time PCR-based method for the quantification of 20 dominant bacterial species and groups of the intestinal flora has been established on the basis of 16S ribosomal DNA taxonomy. A PCR with conserved primers was used for all reactions. In each real-time PCR, a universal probe for quantification of total bacteria and a specific probe for the species in question were included. PCR with conserved primers and the universal probe for total bacteria allowed relative and absolute quantification. Minor groove binder probes increased the sensitivity of the assays 10- to 100-fold. The method was evaluated by cross-reaction experiments and quantification of bacteria in complex clinical samples from healthy patients. A sensitivity of 101 to 103 bacterial cells per sample was achieved. No significant cross-reaction was observed. The real-time PCR assays presented may facilitate understanding of the intestinal bacterial flora through a normalized global estimation of the major contributing species.

* Corresponding author. Mailing address: I. Medical Department, University Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstr. 12, 24105 Kiel, Germany. Phone: 49 431 597 2350. Fax: 49 431 597 1842. E-mail: s.schreiber{at}mucosa.de.

{dagger} S.J.O. and M.M. contributed equally to this study.

Journal of Clinical Microbiology, June 2004, p. 2566-2572, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2566-2572.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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