Development of rapid DNA diagnosis system of poisonous mushrooms

1.为了开发一种新的获得菌根真菌Lyophyllum shimeji单核体的方法，利用12个双核砧木进行了原生质体形成和再生的双核茎培养的单核化。综上所述，我们成功地利用原生质单核化技术制备了单核细胞系，这一技术可用于L食用菌的生物种类鉴定。用UPGMA方法对V4结构域序列进行的分支分析表明，本研究中的12株Lyophyllum shimeji菌株和2株L.decastes菌株被分成两个类群。在交配亲和性试验中，以双核体为原料，通过原生质体单核化制备单核体。根据1类和2类单核群体的交配亲和性试验，表明类1菌株的生物学种类不同。从属于第2类群的菌株中分离出来。我们认为本研究所测试的志贺氏菌株可能是。应包含属于不同生物物种的菌株。以新鲜、烘烤、炒制、天妇罗式和水煮子实体、热干香菇和新鲜、罐头和蒸煮双孢菇的基因组DNA为模板进行了PCR反应。关于。水煮蘑菇、罐头蘑菇和罐头蘑菇的基因组DNA均能扩增出350bp和250bp的条带。因此，DNA诊断技术应用于香菇的种类鉴定是可能的。使用实时聚合酶链式反应系统对日本主要的煮熟的和新鲜的有毒蘑菇进行物种特异性鉴定。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小时内开发出了有毒蘑菇的物种特异性检测方法。

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.