Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood.
We developed a microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species. Nucleotide sequences derived from the internal transcribed spacer (ITS) region of fungal rDNA were used to develop species-specific oligonucleotide probes for Candida albicans, C. tropicalis, C. parapsilosis, and C. krusei. No cross-hybridization was detected with any other fungal, bacterial, or human DNAs tested. In contrast, a C. (Torulopsis) glabrata probe cross-reacted with Saccharomyces cerevisiae DNA but with no other DNAs tested. Genomic DNA purified from C. albicans blastoconidia suspended in blood was amplified by PCR with fungus-specific universal primers ITS3 and ITS4. With the C. albicans-specific probe labeled with digoxigenin, a biotinylated capture probe, and streptavidin-coated microtitration plates, amplified DNA from a few as two C. albicans cells per 0.2 ml of blood could be detected by enzyme immunoassay.
Fujita
S
S
Central Clinical Laboratory, Kanazawa University Hospital, Kanazawa, Japan.
Lasker
B A
BA
Lott
T J
TJ
Reiss
E
E
Morrison
C J
CJ
D000818:Animals; D001483:Base Sequence; D002175:Candida / Q000235:genetics* / Q000302:isolation & purification*; D002177:Candidiasis / Q000175:diagnosis; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000097:blood* / Q000235:genetics*; D004275:DNA, Ribosomal / Q000097:blood / Q000235:genetics; D004076:Digoxigenin; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D005069:Evaluation Studies as Topic; D006801:Humans; D007124:Immunoenzyme Techniques / Q000706:statistics & numerical data; D015336:Molecular Probe Techniques / Q000706:statistics & numerical data; D008969:Molecular Sequence Data; D009172:Mycology / Q000379:methods / Q000706:statistics & numerical data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011817:Rabbits; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D004996:Ethidium; D004076:Digoxigenin