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Multi-probe Real-Time PCR Identification of Four Common Candida Species in Blood Culture Broth

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Abstract

We developed a single-tube real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting Candida albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Primers were designed to amplify 18S rRNA gene of the genus Candida, and DNA probes were designed to hybridize two areas of the amplicons. The amplification curves and specific melting peaks of the probes hybridized with PCR product were used for definite species identifications. The reaction specificity was 100 % when evaluating the assay using DNA samples from 21 isolates of fungal and bacterial species. The assay was further evaluated in 129 fungal blood culture broth samples which were culture positive for fungus. Of the 129 samples, 119 were positively identified as: C. albicans (39), C. tropicalis (30), C. parapsilosis (23), C. glabrata (20), Candida spp. (5), and two samples containing mixed C. glabrata/C. albicans and C. glabrata/C. tropicalis. The five Candida spp. were identified by sequencing analysis as C. krusei, C. dubliniensis, C. aquaetextoris, and two isolates of C. athensensis. Of the ten samples which showed negative PCR results, six were Cryptococcus neoformans, and the others were Trichosporon sp., Rhodotorula sp., Fusarium sp., and Penicillium marneffei. Our findings show that the assay was highly effective in identifying the four medically important Candida species. The results can be available within 3 h after positivity of a blood culture broth sample.

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Acknowledgments

This work was supported by the Siriraj Research Development Fund, Mahidol University. S. Foongladda and M. Chayakulkeeree were supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University.

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Authors and Affiliations

  1. Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Suporn Foongladda, Nanthanida Mongkol & Pornphan Petlum

  2. Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Rd., Bangkoknoi, Bangkok, 10700, Thailand

    Methee Chayakulkeeree

Authors
  1. Suporn Foongladda
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  2. Nanthanida Mongkol
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  3. Pornphan Petlum
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  4. Methee Chayakulkeeree
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Correspondence to Methee Chayakulkeeree.

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Foongladda, S., Mongkol, N., Petlum, P. et al. Multi-probe Real-Time PCR Identification of Four Common Candida Species in Blood Culture Broth. Mycopathologia 177, 251–261 (2014). https://doi.org/10.1007/s11046-014-9743-7

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  • DOI: https://doi.org/10.1007/s11046-014-9743-7

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