title: Development of a multiple detection technique for fungi by DNA microarray with the simultaneous use of internal transcribed spacer region of ribosomal RNA gene and β-tubulin gene probes.
abstract: We offer the first description of the development of a multiple detection technique for fungi by DNA microarray with the simultaneous use of internal transcribed spacer region (ITS) of ribosomal RNA gene and β-tubulin gene probes. The assay uses 12 oligonucleotide probes and multiplex amplification to detect fungal species belonging to various sections of Aspergillus, the Eurotium genus, and the Penicillium genus. The specificity of each probe was tested using 231 reference fungal strains, including 79 target and 152 non-target strains in 102 species of 24 genera. We determined the optimum concentration of the primer pairs for multiplex PCR to be 0.5 μM for the β-tubulin gene and 0.125 μM for the ITS region. In the field trial using 76 specimens containing 323 fungi (up to five fungal strains were included in one specimen), the concordance rate between the DNA microarray and the DNA sequencing results was 97.4％ at the species or genus levels.
authors.affiliation: Corporate Research and Development Toyo Seikan Group Holdings Ltd.
authors.forename: Atsunori
authors.initials: A
authors.lastname: Isshiki
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Takeharu
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Aoki
authors.forename: Mami
authors.initials: M
authors.lastname: Kokaji
authors.forename: Suguru
authors.initials: S
authors.lastname: Tanabe
authors.forename: Taisuke
authors.initials: T
authors.lastname: Kasetani
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Yoshida
mesh_terms: D001230:Aspergillus / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D055325:Eurotium / Q000235:genetics / Q000302:isolation & purification*; D008828:Microbiological Techniques / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D010407:Penicillium / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014404:Tubulin / Q000235:genetics*
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D015345:Oligonucleotide Probes; D014404:Tubulin