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Development of rapid DNA diagnosis system of poisonous mushrooms

毒蘑菇DNA快速诊断系统的研制

基本信息

批准号：
18580164
负责人：
AIMI Tadanori
金额：
$ 2.37万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580164/
关键词：
Poisonous mushroom DNA diagnosis morphology cooking mushroom species DNA data base PCR mushroom identification

项目摘要

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.

1. 为了建立一种获得菌根真菌Lyophyllum shimeji单核细胞的新方法，以12个双核砧木为材料，通过原生质体的形成和再生，进行了双核砧木培养的单核化。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砧木，该技术可用于鉴定石鸡2的生物种类。采用UPGMA方法对12株Lyophyllum shimeji菌株和2株L. decdeces菌株的V4结构域序列进行分支分析，结果表明，本研究的Lyophyllum shimeji菌株和2株Lyophyllum decdeces菌株分为2个聚类。为了进行配偶试验，采用原生质体单核化法制备了单核砧木。根据聚类1和聚类2单核种群间的交配配伍试验，认为聚类1菌株的生物种类是不同的。2株属于聚类2。我们认为，本研究检测的shimeji L.菌株可能。它包含了属于不同生物种类的菌株。从新鲜、烘烤、炒、天妇罗和煮熟的肉和热干香菇的子实体以及新鲜、罐装和蒸煮的双孢蘑菇中分离基因组dna作为PCR反应的模板。蒸煮120分钟以上的香菇、罐装香菇和蒸煮香菇的基因组DNA分别扩增出约350 bp和250 bp的片段。因此，DNA诊断法有可能应用于鸡蛋菇的种类鉴定。利用实时PCR系统对日本主要的熟毒蘑菇和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小时内开发了有毒蘑菇的物种特异性测试。