Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis

Abstract

A single primer pair amplifying a cytochrome P-450 lanosterol-14␣-demethylase (L1A1) gene fragment that encodes a highly conserved region was used to detect yeast DNA in clinical specimens. Positive PCR products were obtained from genomic DNAs of Candida albicans, C. parapsilosis, C. tropicalis, C. guilliermondii, C. krusei, C. (Torulopsis) glabrata, and C. kefyr. No human, bacterial, or parasitic DNA was amplified. The sensitivity was evaluated for C. albicans genomic DNA by using various DNA concentrations (200 pg to 2 fg). The amplified DNAs of Candida species with unknown P-450 L1A1 gene sequences were subcloned and sequenced. Identification at the species level was achieved by digestion of the PCR products with different restriction enzymes. A specific restriction enzyme analysis pattern was determined for each species investigated. Subsequently, we used PCR to detect specific yeast DNA directly with clinical specimens such as blood and bronchoalveolar lavage specimens. After appropriate treatment, the specimens were processed by PCR and the results were compared with those obtained by traditional diagnostic procedures such as cultures and serology. Although preliminary, the PCR results seem to correlate well, at least for blood, with those of antigen detection assays and traditional blood cultures, with a better and earlier detection of candidemia.

Aspergillus PCR testing: results from a prospective PCR study within the AmBiLoad trial

“…The 459 samples consisted of 454 blood samples, three BAL samples, one bronchial aspirate and one muscle biopsy specimen. The median number of samples investigated per patient was 4 (range [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. In 83 patients, more than one sample was investigated.…”

Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR

“…Real-time PCR has been used successfully to diagnose infections that are difficult to detect by conventional culture [23,[25][26][27][28][29][30]. Various molecular approaches have been used for the diagnosis of invasive aspergillosis and candidiasis, e.g., PCR amplification followed by restriction enzyme analysis using species-specific probes, and nested PCR amplification [17][18][19][20]. However, some of these methods detect only Aspergillus spp.…”

“…New rapid methods that can detect IFI early in the course of disease, with high sensitivity and specificity, are thus required. More recently, PCR protocols for diagnosing fungal infections have been described [16][17][18][19][20][21][22]. Molecular diagnostic methods using uni-versal fungal PCR primers and species-specific probes have been developed and evaluated for the detection of fungal DNA in clinical specimens.…”

Mycotic keratitis: an overview of diagnosis and therapy

“…Fluorescent in situ hybridization (FISH), restriction fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE), single stranded conformation polymorphism (SSCP), capillary electrophoresis, nested PCR, MicroSeq D2 large subunit ribosomal DNA sequencing kit, line probe assay (LiPA), and many more methods are being used for fungal species identification. 107,111,[146][147][148][149][150][151][152][153][154][155][156][157][158] All these methods either target diversity in appearance of particular sequence by restriction enzyme ⁄ small random primers or secondary structure obtained by single strand because of interaction between bases. The most important requirement of the method is the analysis of outcome.…”