Reverse cross blot hybridization assay for rapid detection of PCR-amplified DNA from Candida species, Cryptococcus neoformans, and Saccharomyces cerevisiae in clinical samples

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),,ve amplified a fragm ent of the ERG11 gene for cytochrome P-450 lanosterol 14 alpha-demethylase, a crucial enzyme in the biosynthesis of ergosterol, The PCR product was an alyzed in a reverse cross blot hybridization assay with species-specific pr obes directed to a target region of the ERG11 gene of Candida albicans (pCa l), C, guilliermondii (pGui), C, (Torulopsis) glabrata (pGla), C, kefyr (pK ef), C, krusei (pKru), C, parapsilosis (pPar), C, tropicalis (pTro), the ne wly described species C, dubliniensis (pDub), Saccharomyces cerevisiae (pSc e), and Cryptococcus neoformans (pCry), The PCR-reverse cross blot hybridiz ation assay correctly identified multiple isolates of each species tested. No cross-hybridization was detected with any other fungal, bacteria, or hum an DNAs tested. The method was tested against conventional identification o n 140 different clinical samples, including blood and cerebrospinal fluid! from patients with suspected fungal infections. The results agreed with tho se 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 positi vity-culture negativity was justified by clinical findings). Species identi fication time was reduced from a mean of 4 days with conventional identific ation to 7 h with the molecular method. The PCR-reverse cross blot hybridiz ation assay is a rapid method for the direct detection and identification o f yeasts in clinical samples.