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Journal of Clinical Microbiology, December 2002, p. 4423-4427, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4423-4427.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Quantification of Bacteria Adherent to Gastrointestinal Mucosa by Real-Time PCR

Xander W. Huijsdens,1 Ronald K. Linskens,2 Mariëtte Mak,1 Stephan G. M. Meuwissen,2 Christina M. J. E. Vandenbroucke-Grauls,1 and Paul H. M. Savelkoul1*

Departments of Medical Microbiology and Infection Control,1 Gastroenterology, VU University Medical Center Amsterdam, Amsterdam, The Netherlands2

Received 24 April 2002/ Returned for modification 3 July 2002/ Accepted 8 September 2002

The use of real-time quantitative PCR (5' nuclease PCR assay) as a tool to study the gastrointestinal microflora that adheres to the colonic mucosa was evaluated. We developed primers and probes based on the 16S ribosomal DNA gene sequences for the detection of Escherichia coli and Bacteroides vulgatus. DNA was isolated from pure cultures and from gut biopsy specimens and quantified by the 5' nuclease PCR assay. The assay showed a very high sensitivity: as little as 1 CFU of E. coli and 9 CFU of B. vulgatus could be detected. The specificities of the primer-probe combinations were evaluated with samples that were spiked with the species most closely related to E. coli and B. vulgatus and with eight other gut microflora species. Mucosal samples spiked with known amounts of E. coli or B. vulgatus DNA showed no PCR inhibition. We conclude that the 5' nuclease PCR assay may be a useful alternative to conventional culture techniques to study the actual in vivo composition of a complex microbial community like the gut microflora.

* Corresponding author. Mailing address: Department of Medical Microbiology and Infection Control, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. Phone: 31 20 4440552. Fax: 31 20 4440473. E-mail: P.Savelkoul{at}VUmc.nl.

Journal of Clinical Microbiology, December 2002, p. 4423-4427, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4423-4427.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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