一种基于新型RNA底物的蓖麻毒素体外脱嘌呤活性检测方法及应用  

An in Vitro Detection Method for Depurination Activity of Ricin Based on A Novel RNA Substrate and Its Application

作　　者：梁龙辉 程茜 杨旸 闫珑 于惠兰 杜斌 刘昌财 刘石磊 LIANG Long-Hui;CHENG Xi;YANG Yang;YAN Long;YU Hui-Lan;DU-Bin;LIU Chang-Cai;LIU Shi-Lei(State Key Laboratory of NBC Protection for Civilian,Beijing 102205,China;College of Liberal Arts and Sciences,National University of Defense Technology,Changsha 410073,China)

机构地区：[1]国民核生化灾害防护国家重点实验室,北京102205 [2]国防科技大学文理学院,长沙410073

出　　处：《分析化学》2021年第10期1694-1703,I0017-I0019,共13页Chinese Journal of Analytical Chemistry

基　　金：国民核生化灾害防护国家重点实验室项目(No.SKLNBC2020-08)资助。

摘　　要：建立了一种基于新型RNA底物脱嘌呤的蓖麻毒素(Ricin)体外活性检测方法,解决了传统Ricin体外脱嘌呤活性检测方法中底物易发生自水解而产生假阳性结果的问题。比较了Ricin脱嘌呤体外活性检测中普遍使用的单链DNA及相同序列RNA底物的活性差异,证实了RNA比DNA更适于作为Ricin脱嘌呤反应底物。对脱嘌呤反应条件进行优化,发现pH值、温度和RNA底物浓度是影响脱嘌呤反应效率的关键因素,在优化条件下,阴性对照样品中无腺嘌呤干扰。进一步对系列RNA底物进行系统筛选,总结了RNA茎-环结构组成与底物活性的规律,筛选得到了一种用于Ricin体外脱嘌呤活性检测的新型RNA底物,其活性是已报道最优底物的2倍。最终,基于筛选出的新型RNA底物,结合免疫磁珠特异性亲和方法,建立了复杂基质中活性Ricin的同位素标记腺嘌呤内标-液相色谱-三重四极杆质谱多反应监测模式定量检测方法。本方法专属性好,Ricin体外脱嘌呤活性检测的线性范围为5.0~300 ng/mL,检出限为1.0 ng/mL(S/N=3)。将本方法应用于自来水、牛奶和血浆样品中活性Ricin的定量检测,加标回收率在91.0%~116.5%之间,相对标准偏差在3.2%~9.3%之间。本研究为食品安全以及化学武器核查等领域中活性Ricin的筛查鉴定提供了有效方法。An in vitro detection method for active ricin based on depurination of a novel RNA substrate was developed with no adenine interference in the negative control sample.The problem that the substrate was prone to self-hydrolysis and caused false positive results in the traditional depurination activity detection method was solved.The depurination activities of single-stranded DNA and RNA substrates with the same sequence were systematically compared.It was found that RNA was more suitable as the substrate for detection of ricin depurination activity.The optimization of reaction conditions showed that the pH values,temperature,and concentration of RNA substrate were the key factors for detection of ricin depurination activity.Under the optimal conditions,there was no adenine interference in the negative control sample.The influence of RNA stem-loop composition on substrate activity was validated by screening a series of RNA substrates.A novel RNA substrate was obtained with the optimal depurination activity,and its depurination activity was twice that of the reported substrate.By combining with the immunocapture purification,a quantitative detection method for the active ricin by isotope-labeled internal liquid chromatography-triple quadrupole mass spectrometry with multiple reaction monitoring using the novel RNA as the depurination substrate was established.This method had good specificity,the linear range of active ricin ranged from 5.0 to 300 ng/mL,and the limit of detection was 1.0 ng/mL(S/N=3).This method was applied to the quantitative detection of active ricin in tap water,milk,and plasma samples.The recoveries of spiked samples were between 91.0% and 116.5%,and the relative standard deviations(RSDs)were between 3.2% and 9.3%.This method was helpful for the screening and identification of active ricin in the fields of food safety and chemical weapons inspection.

关 键 词：蓖麻毒素 RNA底物 脱嘌呤活性 Ⅱ型核糖体失活蛋白 多反应监测模式 

分 类 号：TS207.3[轻工技术与工程—食品科学] Q503[轻工技术与工程—食品科学与工程] O657.63[生物学—生物化学]