Multiplex Real-Time PCR Assay for Simultaneous Detection of Omphalotus guepiniformis and Lentinula edodes

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  • TSURUDA Sayuri
    Fukuoka City Institute for Hygiene and the Environment Fukuoka City Institute for Hygiene and the Environment
  • AKAKI Kouichi
    Fukuoka City Institute for Hygiene and the Environment Fukuoka City Institute for Hygiene and the Environment
  • HIWAKI Hiroshi
    Fukuoka City Institute for Hygiene and the Environment Fukuoka City Institute for Hygiene and the Environment
  • SUZUKI Akira
    Faculty of Education, Chiba University Faculty of Education, Chiba University
  • AKIYAMA Hiroshi
    National Institute of Health Sciences National Institute of Health Sciences

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  • Multiplex Real-Time PCR Assay for Simultaneous Detection of <i>Omphalotus guepiniformis</i> and <i>Lentinula edodes</i>

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A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.

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