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Journal of Clinical Microbiology, March 2007, p. 874-880, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01556-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Multiplex Real-Time PCR Targeting the RNase P RNA Gene for Detection and Identification of Candida Species in Blood{triangledown}

Åsa Innings,1 Måns Ullberg,1 Anders Johansson,1,2 Carl Johan Rubin,1 Niklas Noreus,1 Magnus Isaksson,1 and Björn Herrmann1*

Department of Clinical Microbiology, Uppsala University Hospital, Uppsala, Sweden,1 Department of Molecular and Clinical Medicine, Linköping University, Linköping, Sweden2

Received 27 July 2006/ Returned for modification 11 September 2006/ Accepted 26 December 2006

We have developed a single-tube multiplex real-time PCR method for the detection of the eight most common Candida species causing septicemia: Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis. The method developed targets the RNase P RNA gene RPR1. Sequences of this gene were determined for seven of the Candida species and showed surprisingly large sequence variation. C. glabrata was found to have a gene that was five times longer gene than those of the other species, and the nucleotide sequence similarity between C. krusei and C. albicans was as low as 55%. The multiplex PCR contained three probes that enabled the specific detection of C. albicans, C. glabrata, and C. krusei and a fourth probe that allowed the general detection of the remaining species. The method was able to detect 1 to 10 genome copies when the detection limit was tested repeatedly for the four species C. albicans, C. glabrata, C. krusei, and C. guilliermondii. No significant difference in the detection limit was seen when the multiplex format was compared with single-species PCR, i.e., two primers and one probe. The method detected eight clinically relevant Candida species and did not react with other tested non-Candida species or human DNA. The assay was applied to 20 blood samples from nine patients and showed a sensitivity similar to that of culture.

* Corresponding author. Mailing address: Department of Clinical Microbiology, Uppsala University Hospital, SE-751 85 Uppsala, Sweden. Phone: 46 18 6113952. Fax: 46 18 559157. E-mail: bjorn.herrmann{at}medsci.uu.se.

{triangledown} Published ahead of print on 10 January 2007.

Journal of Clinical Microbiology, March 2007, p. 874-880, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01556-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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