Development of rapid DNA diagnosis system of poisonous mushrooms

1.为了探索一种获得离褶伞单核体的新方法，以12个离褶伞为材料，通过原生质体形成和再生进行了离褶伞培养物的单核化。综上所述，我们成功地通过原生质体单核化技术制备了单核原种，该技术可用于鉴定Lshimeji的生物种. UPGMA方法对V4结构域序列进行分支分析，结果表明12个离褶伞菌株和2个离褶伞菌株属于同一种。在本研究中测试的Decastes菌株分为两个簇。对于交配相容性试验，通过原生质体单核化从双核体储备物制备单核体储备物。根据群1和群2的单核系交配亲和性试验结果，群1菌株的生物学种可能不同。与属于聚类2的菌株相比。我们建议L.本研究中测试的Shimeji菌株可能。含有不同生物种的菌株.以新鲜、烘烤、清炒、天妇罗式和水煮的香菇子实体、热干香菇子实体和新鲜、罐装和蒸煮的双孢蘑菇子实体的基因组DNA为模板进行PCR反应。关于.蒸煮120 min以上的香菇、罐头和蒸煮香菇的基因组DNA分别扩增出350 bp和250 bp的片段。因此，DNA诊断技术有可能应用于平菇的种属鉴定.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种属特异性鉴定。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.