含鹅膏环肽剧毒蘑菇的快速检测方法构建及应用

	含鹅膏环肽剧毒蘑菇的快速检测方法构建及应用
	李鑫灿
导师	罗宏
关键词	有毒蘑菇，中毒，鹅膏环肽，湿管法，显色反应 Lethal mushrooms, Mushroom poisoning, Amanita cyclic peptides, The wet-tube test, Chromogenic reaction
摘要	野生蘑菇作为一种无污染的有机食品，因其味道鲜美和营养价值较高而广受世界各国人民的青睐。由于有毒野生蘑菇与可食用在形态结构上极为相似，误食有毒蘑菇引发的中毒事件频发，致死事件发生数量最多的是含有鹅膏毒肽的剧毒蘑菇，占比高达90%以上。鹅膏毒肽为真核生物的RNA聚合酶Ⅱ抑制剂，中毒症状分为潜伏期、胃肠炎期、假愈期和内脏损害期4个阶段，并且由于其在人体内经过肝肠循环而局部聚集，导致难以通过常规方法检测。而且，现有的检测方法对大型仪器依赖性强，不能在现场（野外、市场、营地、卫生所等）使用，无法满足方便、快捷鉴定毒素的需求。据此，本论文总结前人的研究经验（主要为Meixner test），构建了高效快捷的针对含有鹅膏毒肽剧毒蘑菇及鹅膏毒肽纯毒素的检测方法——湿管法，3分钟即可检测得出结果，可满足多种应用场景。基于此方法，研发出“鹅膏毒素剧毒蘑菇快速检测试剂盒”，为首个检测有毒蘑菇的产品。本论文还确定了湿管法（反应体积为100 μL）的助剂组合：离子助剂为4.8%氯化钾，固体助剂为60 μg/mL粉末A，结果表明以上助剂对显色具有显著增强作用。本论文构建的含鹅膏环肽剧毒蘑菇的快速检测方法及其应用体系，对误食有毒野生蘑菇中毒的处置与救治提供了高效快捷的技术手段。主要结果如下： 1．构建了高效快捷的鹅膏毒肽检测方法——“湿管法”。该方法彻底改良了麦氏显色法，所具备的新特点为：（1）灵敏度高，微量样品。20 ug反应浓度为200 μg/mL的纯毒素即可满足检测要求；（2）极大地缩短了反应时间，3 min既可观察显色结果；（3）湿管法完全摆脱了干燥步骤，反应在潮湿环境（离心管）中迅速完成；（4）极高的检测特异性，与鬼笔毒肽（phalloidin）等无假阳性反应；（5）盐酸浓度降低至15%，确保操作安全的同时保证了显色效果。 2．研发出“鹅膏毒素剧毒蘑菇快速检测试剂盒”，为首个检测有毒蘑菇的产品。该产品的特点及优势为——微样品、高精度、快结果：（1）微量样品（黄豆大小子实体或20 μg纯毒素）即可被试剂盒测出；（2）对鹅膏毒素剧毒蘑菇检测具有极高灵敏度，反应会出现特征性的蓝绿色（或绿色）；（3）试剂盒可在3–5分钟内即可得到显色结果，提高了检测效率。试剂盒对于实施有毒蘑菇的中毒防控与治疗具有现实意义。 3．确定了湿管法的有效助剂组合。本论文完成了对120种有机物和无机物的筛选，有机物中：乙醇、异戊醇、苯、乙酸乙酯、柠檬酸、水杨酸、乙酸铵对显色反应有一定的增强作用，无机物中：氯化镁、氯化锌、氯化镓、氯化铷、氯化铟、氯化钡、氯化镧、氯化钆、氯化铽、氯化镝、氯化铅、氯化铱、氢氟酸、氯化铵、过硫酸钾、磷酸钾对显色反应有一定的增强作用。但是上述物质的显色作用明显不如氯化钾，因此最终确定助剂组合为：离子助剂为4.8%氯化钾，固体助剂为60 μg/mL粉末A。上述助剂组合对增强显色具有明显作用，可在试剂盒后期改良中加以应用。; As a non-polluting organic food, wild mushroom is widely favored by people all over the world because of its delicious taste and high nutritional value. However, lethal mushrooms can be very similar to edible ones in morphology and structure, frequently causing poisoning events. The most severe events are caused by the lethal mushrooms containing lethal Amanita cyclic peptides, accounting for more than 90% of all deadly cases. Lethal Amanita cyclic peptides are inhibitors of RNA polymerase Ⅱ in eukaryotes. The symptoms of poisoning are divided into four stages: incubation period, gastroenteritis period, latent phase and liver damage stage. It is difficult to be detected by conventional methods because it accumulates locally through hepatoenteric circulation in human body. In addition, the existing detection methods are highly dependent on large instruments and cannot be used in on site (fields, markets, camps and health posts), and cannot meet the needs of convenient and fast identification of toxins. Based on previous methods (mainly Meixner test), this study developed an efficient and fast detection method of lethal mushrooms containing Amanita cyclic peptides or the pure toxins, which was named wet-tube test caperbal of detecting the toxins in 3 minutes, in a variety of scenarios. Based on the wet–tube test, this topic developed the Rapid Detection Kit for Amatoxin-containing Mushrooms, which was the first product for toxic mushrooms and have received good feedback. This paper also determined the accessory ingredient combination of wet-tube test (reaction volume is 100 μL): ionic accessory ingredient was 4.8% potassium chloride, and solid accessory ingredient is 60 μg/mL powder A. The results show that the accessory ingredient combination had a significant enhancing effect on chromogenic reaction. The kit has practical significance for the prevention, control and treatment of lethal mushrooms. The main results are as follows: 1. A rapid and efficient method for the determination of Amanita cyclic peptides was established. The wet-tube test absolutely changes the Meixner test, and the characteristics of this method are as follows: (1) The wet-tube test has high sensitivity, and microscale sample of 20 μg pure toxin at 200 μg/mL) are sufficient for the detection. (2) The wet-tube test greatly shortened the reaction time only 3 min for the chromogenic reaction to complete. (3) The wet-tube test completely avoided the desiccation steps required in Meixner test, and all operations are now completed in an Eppendorf tube. (4) The specificity of the wet-tube test was improved, and there was no false positive reaction with phalloidin. (5) The applied concentration of hydrochloric acid was reduced to 15%, and the heating temperature was 70 ℃, ensuring the safety of operation and the coloration effect. 2. Developed Rapid Detection Kit for Amatoxin-containing Mushrooms, the first product to detect poisonous mushrooms. The characteristics and advantages
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75163
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	李鑫灿. 含鹅膏环肽剧毒蘑菇的快速检测方法构建及应用[D]. 中国科学院大学,2022.

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