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Journal of Clinical Microbiology, February 2004, p. 512-517, Vol. 42, No. 2
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.2.512-517.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Detection and Differentiation of In Vitro-Spiked Bacteria by Real-Time PCR and Melting-Curve Analysis

S. Klaschik,1* L. E. Lehmann,1 A. Raadts,1 M. Book,1 J. Gebel,2 A. Hoeft,1 and F. Stuber1

Department of Anesthesiology and Intensive Care Medicine,1 Institute for Hygiene and Public Health, University of Bonn, Bonn, Germany2

Received 6 March 2003/ Returned for modification 14 April 2003/ Accepted 30 September 2003

We introduce a consensus real-time PCR protocol for the detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and plasma. This prototype detection system enables an exact Gram stain classification and, in particular, screening for specific species of 17 intensive care unit-relevant bacteria by means of fluorescence hybridization probes and melting-curve analysis in a one-run experiment. One strain of every species was tested at a final density of 106 CFU/ml. All bacteria examined except Staphylococcus aureus and Staphylococcus epidermidis could be differentiated successfully; S. aureus and S. epidermidis could only be classified as "Staphylococcus species." The hands-on time for preparation of the DNA, performance of the PCR, and evaluation of the PCR results was less than 4 h. Nevertheless, this prototype detection system requires more clinical validation.

* Corresponding author. Mailing address: Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Phone: 0049-228-287-6018. Fax: 0049-228-287-6754. E-mail: klaschik{at}ukb.uni-bonn.de

Journal of Clinical Microbiology, February 2004, p. 512-517, Vol. 42, No. 2
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.2.512-517.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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