An improved rapid quantitative detection and identification method for a wide range of fungi.
To develop a rapid and quantitative diagnostic technique for the detection and identification of a wide range of fungi, an improved molecular method based on real-time PCR and the analysis of its products that targets the internal transcribed spacer (ITS) 2 region was established. The real-time PCR could quantitatively and specifically detect the ITS2 region from all 24 tested pathogenic fungal species at between 10(1) and 10(7) copies per test without amplification of bacterial or human DNA. The sequences of the primer-binding sites are conserved in the registered sequences of 34 other pathogenic fungal species, suggesting that the PCR would also detect these species. The hyperpolymorphic nature of the ITS2 region between fungal species in terms of length and nucleotide sequence provided valuable information for the determination of species. By labelling the 5' end of the reverse primer with NED fluorescent dye, the fragment lengths of the real-time PCR products and their 3'-terminal fragments, derived using restriction enzyme ScrFI digestion, were easily evaluated by capillary electrophoresis. Using this analysis, the number and species of fungi present in samples could be estimated. Moreover, sequence analysis of the real-time PCR products could accurately determine species in samples containing a single species. This diagnostic technique can estimate a wide range of fungi from various clinical samples within 1 day and accurately identify them in 2 days. Quantitative results for fungal titre in samples can also provide useful information for understanding the progression of disease and the efficacy of antifungal chemotherapy.
Soeta
Nobutoshi
N
Department of Surgery I, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.
Terashima
Masanori
M
Department of Surgery I, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.
Gotoh
Mitsukazu
M
Department of Surgery I, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.
Mori
Shuichi
S
Department of Microbiology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.
Nishiyama
Kyoko
K
Department of Microbiology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.
Ishioka
Ken
K
Department of Microbiology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.
Kaneko
Hisatoshi
H
Department of Microbiology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.
Suzutani
Tatsuo
T
Department of Microbiology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.
D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D005658:Fungi / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D021903:DNA, Ribosomal Spacer; D012337:RNA, Ribosomal, 18S