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This Article Full Text (Papers in Press[PDF]) All Versions of this Article: jmm.0.006858-0v1 58/8/1037    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Google Scholar Articles by Soeta, N. Articles by Suzutani, T. PubMed PubMed Citation Articles by Soeta, N. Articles by Suzutani, T. Agricola Articles by Soeta, N. Articles by Suzutani, T.

An improved rapid quantitative detection and identification method for a wide range of fungi

¹ Southern Tohoku Research Institute for Neuroscience;

² Shizuoka Cancer Center;

³ Fukushima Medical University School of Medicine

⁴ E-mail: suzutani{at}fmu.ac.jp

Received September 23, 2008
Accepted May 4, 2009

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 can quantitatively and specifically detect the ITS2 region from all 24 tested pathogenic fungal species at between 101 to 107 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 hyper-polymorphic nature of the ITS2 region between fungal species in terms of length and nucleotide sequence provided valuable information for the determination of species. By labeling the 5' end of the reverse primer with NED fluorescent dye, the fragment length of the real-time PCR products and their 3'-terminal fragments, derived using restriction enzyme Scr FI 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. Therefore, 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 titer in samples can also provide useful information for understanding the progression of disease and the efficacy of antifungal chemotherapy.