Identification of rare Candida species and other yeasts by polymerase chain reaction and slot blot hybridization.
Invasive candidiasis has become a major cause of morbidity and mortality in immunocompromised hosts. Here we describe a fast and reliable DNA extraction and PCR amplification method in combination with a slot blot hybridization assay. A genus-specific probe was designed that allowed to detect DNA from a broad range of Candida species and 3 other yeasts. In addition, species-specific oligonucleotides for emerging Candida and other yeast species allowed to identify DNA extracted from Candida lusitaniae, Candida humicola, Candida kefyr, Candida inconspicua, Candida solani, Malassezia furfur and Trichosporon cutaneum. A sensitivity of at least 10(1) CFU, corresponding to 100 fg of fungal DNA, was documented for all species-specific probes and the common Candida probe. In addition, the 18S rRNA genes of 7 yeast species (C. humicola, C. kefyr, C. solani, C. inconspicua, C. norvegensis, C. utilis and M. furfur) were completely sequenced. The sequencing primers described bind to highly conserved primer binding sites. Therefore, these primers would allow rapid cycle sequence of additional ribosomal genes throughout the whole kingdom of fungi.
Loeffler
J
J
Eberhard-Karls-Universität Tübingen, Medizinische Klinik, Abteilung II, Tuebingen, Germany. juergen.loeffler@med.uni-tuebingen.de
Hebart
H
H
Magga
S
S
Schmidt
D
D
Klingspor
L
L
Tollemar
J
J
Schumacher
U
U
Einsele
H
H
D002175:Candida / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D002177:Candidiasis / Q000382:microbiology*; D004271:DNA, Fungal / Q000032:analysis* / Q000235:genetics; D020459:Genes, rRNA; D006801:Humans; D008290:Malassezia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D003904:Mitosporic Fungi / Q000145:classification*; D008969:Molecular Sequence Data; D009181:Mycoses / Q000382:microbiology; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014250:Trichosporon / Q000145:classification / Q000235:genetics / Q000302:isolation & purification
D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S