Development of rapid DNA diagnosis system of poisonous mushrooms

1. 为了开发一种获得菌根真菌（Lyophyllum shimeji）单核的新方法，使用 12 个双核菌种通过原生质体形成和再生对双核 stodc 培养物进行单核化。总之，我们通过原生质体单核化成功地制备了单核细胞，该技术可用于鉴定 L shimeji 的生物物种。 2．通过 UPGMA 方法对 V4 结构域序列进行的分支分析表明，本研究中测试的 12 种真姬菇菌株和 2 种真姬菇菌株分为两个簇。对于交配相容性测试，通过原生质体单核化从双核原种制备单核原种的制剂。根据簇1和簇2的单核种群之间的交配亲和性测试，表明簇1的菌株的生物种类不同。与属于簇 2 的菌株相比。我们认为本研究中测试的 L. shimeji 菌株可能是这样。含有属于不同生物物种的菌株。3．从新鲜、烘烤、炒、天妇罗和煮的肉质子实体和热干香菇以及新鲜、罐装和蒸煮双孢蘑菇中分离的基因组 DNA 用作 PCR 反应的模板。关于。煮蘑菇、罐装蘑菇和蒸煮蘑菇的基因组 DNA 分别经过 120 分钟以上即可扩增出 350 bp 和 250 bp 的片段。因此，DNA诊断技术有可能适用于翘起蘑菇的物种鉴定。4．使用实时 PCR 系统对日本主要的熟毒蘑菇和新鲜毒蘑菇进行了物种特异性鉴定。检测到特异性荧光信号，未检测到非特异性信号。因此，我们成功地在1.5小时内开发出了针对有毒蘑菇的物种特异性测试。

1. In order to develop a novel method of obtaining monokaryons for a mycorrhizal fiungus, Lyophyllum shimeji, monokaryotization of dikaryotic stodc culture via protoplast formation and regeneration was performed using 12 dikaryotic stocks. In conclusion, we successfully prepared monokaryotic stocks via protoplast monookaryotization, a technique that can be used to identify biological species of L shimeji.2. The cladistic analysis of the V4 domain sequences, performed by UPGMA methods, revealed that the twelve Lyophyllum shimeji strains and two L. decastes strains were tested in this study divided into two clusters. For mating compatibility tests, the preparation of monokaryotic stocks were prepared from dikaryon stocks by protoplast monokaryotization. According to mating compatibility tests between monokaryotic stocks belong to cluster 1 and 2, it is suggested that the biological species of strains belong to cluster 1 are different. from that of strains belong to cluster 2. We suggest that the L. shimeji strains tested in this study might. be contained the strains belonging to different biological species.3. Genomic DNAs isolated from fresh, baked, stir-fried, tempura-style and boiled fruiting bodies of flesh and heat-dry Lentinula edodes and fresh, canned and retorted Agaricus bisporus were used as templates for PCR reactions. About. 350 bp and 250 bp fragments could be amplified from genomic DNA of boiled mushroom for more than 120 minutes, and that of canned and retorted mushroom, respectively. Therefore, it is possible that DNA diagnostics is applicable for species identification of cocked mushroom.4. Species-specific identification of the cooked and fresh poisonous mushrooms that are major in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no non-specific signals were detected. Therefore, we succeeded in developing a species-specific test for poisonous mushrooms within 1.5 hours.