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Journal of Clinical Microbiology, September 2003, p. 4016-4021, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4016-4021.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Development and Clinical Evaluation of an Internally Controlled, Single-Tube Multiplex Real-Time PCR Assay for Detection of Legionella pneumophila and Other Legionella Species

Kate E. Templeton,1 Sitha A. Scheltinga,1 Peter Sillekens,2 Jantine W. Crielaard,1 Alje P. van Dam,1 Herman Goossens,1,3 and Eric C. J. Claas1*

Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden,1 BioMerieux, Boxtel, The Netherlands,2 Department of Microbiology, University of Antwerp, Antwerp, Belgium3

Received 3 March 2003/ Returned for modification 14 April 2003/ Accepted 27 May 2003

A multiplex real-time PCR assay for detection of Legionella pneumophila and Legionella spp. and including an internal control was designed. Legionella species, L. pneumophila, and the internal control were detected simultaneously by probes labeled with 6-carboxy-fluorescein, hexachlorofluorescein, and indodicarbocyanine, respectively. Therefore, no postamplification analysis was required in order to distinguish the targets. The sensitivity of both assays was 2.5 CFU/ml, and from analysis of 10 culture-positive and 74 culture-negative samples from patients investigated for legionellosis, 100% agreement was observed by both assays in comparison to culture. Four additional positives were found by the multiplex real-time PCR assay in the Legionella culture-negative samples.

* Corresponding author. Mailing address: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Phone: 0031 71 526 3650. Fax: 0031 71 524 8148. E-mail: E.C.J.Claas{at}LUMC.NL.

Journal of Clinical Microbiology, September 2003, p. 4016-4021, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4016-4021.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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