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Journal of Clinical Microbiology, April 2000, p. 1609-1614, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Reverse Cross Blot Hybridization Assay for Rapid Detection of PCR-Amplified DNA from Candida Species, Cryptococcus neoformans, and Saccharomyces cerevisiae in Clinical Samples

Brunella Posteraro,1 Maurizio Sanguinetti,1 Luca Masucci,1 Lucio Romano,1 Giulia Morace,2,* and Giovanni Fadda1

Istituto di Microbiologia, Università Cattolica del Sacro Cuore, Rome,1 and Istituto di Microbiologia, Università degli Studi di Milano, Milan,2 Italy

Received 1 October 1999/Returned for modification 3 November 1999/Accepted 16 December 1999

A PCR-based assay was developed to detect and identify medically important yeasts in clinical samples. Using a previously described set of primers (G. Morace et al., J. Clin. Microbiol. 35:667-672, 1997), we amplified a fragment of the ERG11 gene for cytochrome P-450 lanosterol 14alpha -demethylase, a crucial enzyme in the biosynthesis of ergosterol. The PCR product was analyzed in a reverse cross blot hybridization assay with species-specific probes directed to a target region of the ERG11 gene of Candida albicans (pCal), C. guilliermondii (pGui), C. (Torulopsis) glabrata (pGla), C. kefyr (pKef), C. krusei (pKru), C. parapsilosis (pPar), C. tropicalis (pTro), the newly described species C. dubliniensis (pDub), Saccharomyces cerevisiae (pSce), and Cryptococcus neoformans (pCry). The PCR-reverse cross blot hybridization assay correctly identified multiple isolates of each species tested. No cross-hybridization was detected with any other fungal, bacteria, or human DNAs tested. The method was tested against conventional identification on 140 different clinical samples, including blood and cerebrospinal fluid, from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but six specimens (two of which grew yeasts not included in the PCR panel of probes and four in which PCR positivity-culture negativity was justified by clinical findings). Species identification time was reduced from a mean of 4 days with conventional identification to 7 h with the molecular method. The PCR-reverse cross blot hybridization assay is a rapid method for the direct detection and identification of yeasts in clinical samples.

* Corresponding author. Mailing address: Istituto di Microbiologia, Università degli Studi di Milano, Via Pascal, 36, 20133 Milan, Italy. Phone: 39-02-26601215. Fax: 39-02-26601218. E-mail: giulia.morace{at}unimi.it.

Journal of Clinical Microbiology, April 2000, p. 1609-1614, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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