Isshiki Atsunori, Takeharu Hitoshi, Aoki Shunsuke, Kokaji Mami, Tanabe Suguru, Kasetani Taisuke, Yoshida Mitsuhiro
Corporate Research and Development Toyo Seikan Group Holdings Ltd.
Biocontrol Sci. 2014;19(3):139-45. doi: 10.4265/bio.19.139.
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.
我们首次描述了一种利用DNA微阵列同时使用核糖体RNA基因的内部转录间隔区（ITS）和β-微管蛋白基因探针来检测真菌的多重检测技术的开发情况。该检测方法使用12种寡核苷酸探针和多重扩增来检测属于曲霉属各个部分、散囊菌属和青霉属的真菌物种。使用231株参考真菌菌株测试了每种探针的特异性，这些菌株包括24个属102种中的79株靶标菌株和152株非靶标菌株。我们确定多重PCR引物对的最佳浓度对于β-微管蛋白基因为0.5μM，对于ITS区域为0.125μM。在使用包含323种真菌的76个标本（一个标本中最多包含五种真菌菌株）的现场试验中，DNA微阵列和DNA测序结果在物种或属水平上的一致性率为97.4％。
