摘要
目的探究中药材提取物对诺如病毒的灭活作用及其机理,为预防食源性诺如病毒的爆发提供新的思路及依据。方法制备蜂胶醇提物、鱼腥草水提物,用逆转录实时定量PCR法检测病毒的含量研究各提取物对诺如病毒的灭活的量效和时效关系,比较其与紫外线、二氧化氯、双氧水的灭活效果;用酶联免疫吸附测定病毒完整衣壳蛋白的含量以分析提取物对其破坏作用;PCR扩增特异性片段并进行测序分析研究提取物对病毒核酸的破坏,以进一步阐述灭活机理。结果蜂胶醇提物和鱼腥草水提物对诺如病毒有灭活作用,在50mg·mL^-1作用30 min灭活率可达97.03%和97.88%,10 mg·mL^-1二氧化氯作用30 min灭活率85.13%、9 000μW·cm^-2的紫外作用30 min后灭活率96.7%、5%双氧水作用30 min后灭活率81.09%;与对照组相比,鱼腥草水提物、紫外线、二氧化氯和双氧水处理后病毒外壳蛋白表含量明显下降,但是蜂胶醇提物对诺如病毒颗粒的破坏作用不显著。5种方法对病毒核酸均有破坏作用,3个特异性片段鱼腥草组和双氧水组均未能扩增成功,蜂胶组、紫外组仅扩增1个片段,二氧化氯组扩出了2个片段;序列分析显示,开放阅读框(Open Reading Frame,ORF)ORF2和ORF3均未被扩出,蜂胶醇提物与紫外线对诺如病毒核酸突变主要作用于ORF1中VPg、Pro及RdRp蛋白位点;而鱼腥草水提物与双氧水对诺如病毒ORF1核酸无突变效应。结论蜂胶醇提物、鱼腥草水提物对诺如病毒均有较高的灭活率,两者对病毒核酸均有明显的破坏作用,鱼腥草水提物尚能破坏病毒外壳蛋白的结构。
Objective:To explore the inactivation and mechanism of extracts of Chinese herbal medicines on Norovirus,and to provide new ideas and basis for preventing the outbreak of foodborne Norovirus.Methods:Propolis ethanol extract and Houttuynia cordata aqueous extract were prepared.RT-qPCR was used to detect the content of virus to study the timeliness and dose-effect relationship between the two Chinese herbal extracts on the inactivation of Norovirus,and compare the inactivation effect with UV,chlorine dioxide and hydrogen peroxide.The content of viral capsid protein was determined by enzyme-linked immunosorbent assay(ELISA)to analyze the damage effect of extracts.Amplified specific fragments by PCR and sequencing was used to study the damage of the extract to viral nucleic acid,so as to further elucidate the inactivation mechanism.Results:Propolis ethanol extract and Houttuynia cordata aqueous extract had inactivation effect on norovirus,with the inactivation rate of 97.03%and 97.88%at 30 min of 50 mg·mL^-1,10 mg·mL^-1 chlorine dioxide effect 30min inactivation rate 85.13%,9000μW·cm^-2 UV after 30 min inactivation rate 96.7%,5%hydrogen peroxide 30 min after inactivation rate 81.09%;Compared with the control group,the contents of virus shell protein were significantly decreased after treated with Houttuynia cordata aqueous extract,UV,chlorine dioxide and hydrogen peroxide,but the effect of Propolis ethanol extract on Norovirus particles was not significant.All of the five methods had a destructive effect on viral nucleic acid.Three specific fragments of Houttuynia cordata group and hydrogen peroxide group were not successfully amplified.Sequence analysis showed that ORF2 and ORF3 were not expanded,and Propolis ethanol extract and UV were mainly responsible for the mutation of norovirus nucleic acid at the VPg,Pro and RdRp protein sites in ORF1.And there was no mutagenic effect of Houttuynia cordata aqueous extract and hydrogen peroxide on ORF1 nucleic acid of norovirus.Conclusion:Propolis ethanol extract and Hout
作者
王书菁
施晓峰
廖宁波
吴康军
周文婕
卢宇剑
程东庆
Wang Shujing;Shi Xiaofeng;Liao Ningbo;Wu Kangjun;Zhou Wenjie;Lu Yujian;Cheng Dongqing(College of Medical Technology,Zhejiang Chinese Medicine University,Hangzhou 310053,China;Zhejiang Provincial Center for Disease Control and Prevention,Hangzhou 310051,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2019年第6期1167-1172,共6页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
国家自然科学基金委员会青年科学基金项目(31701715):果蔬表面HBGAs表达细菌对诺如病毒存活及持续性感染的影响,负责人:廖宁波
关键词
蜂胶
鱼腥草
诺如病毒
灭活
Propolis
Houttuynia cordata
norovirus
inactivation
