TY - JOUR
T1 - Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis
AU - Morace, Giulia
AU - Sanguinetti, Maurizio
AU - Posteraro, Brunella
AU - Fadda, Giovanni
PY - 1997
Y1 - 1997
N2 - A single primer pair amplifying a cytochrome P-450 lanosterol-14 alpha-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.
AB - A single primer pair amplifying a cytochrome P-450 lanosterol-14 alpha-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.
KW - AIDS-Related Opportunistic Infections
KW - Amino Acid Sequence
KW - Base Sequence
KW - Bronchoalveolar Lavage Fluid
KW - Candida
KW - Candidiasis
KW - Cytochrome P-450 Enzyme System
KW - DNA Primers
KW - DNA Restriction Enzymes
KW - DNA, Fungal
KW - Evaluation Studies as Topic
KW - Fungemia
KW - Genes, Fungal
KW - Humans
KW - Immunocompromised Host
KW - Molecular Sequence Data
KW - Mycology
KW - Opportunistic Infections
KW - Oxidoreductases
KW - Polymerase Chain Reaction
KW - Sensitivity and Specificity
KW - Species Specificity
KW - Sterol 14-Demethylase
KW - AIDS-Related Opportunistic Infections
KW - Amino Acid Sequence
KW - Base Sequence
KW - Bronchoalveolar Lavage Fluid
KW - Candida
KW - Candidiasis
KW - Cytochrome P-450 Enzyme System
KW - DNA Primers
KW - DNA Restriction Enzymes
KW - DNA, Fungal
KW - Evaluation Studies as Topic
KW - Fungemia
KW - Genes, Fungal
KW - Humans
KW - Immunocompromised Host
KW - Molecular Sequence Data
KW - Mycology
KW - Opportunistic Infections
KW - Oxidoreductases
KW - Polymerase Chain Reaction
KW - Sensitivity and Specificity
KW - Species Specificity
KW - Sterol 14-Demethylase
UR - http://hdl.handle.net/10807/17413
U2 - 10.1128/jcm.35.3.667-672.1997
DO - 10.1128/jcm.35.3.667-672.1997
M3 - Article
SN - 0095-1137
VL - 35
SP - 667
EP - 672
JO - Journal of Clinical Microbiology
JF - Journal of Clinical Microbiology
ER -