作者：阮佳1,2, 张薇薇1, 刘鑫1, 苏会岚1
作者单位：1. 成都医学院，四川 成都 621000;
2. 四川大学华西公共卫生学院，四川 成都 610041
关键词：诺如病毒;轮状病毒;莴苣;富集
摘要：
蔬菜被诺如病毒和轮状病毒污染后出现食物中毒的风险高，准确地检测蔬菜中两种食源性病毒对保障食品安全具有重要意义。分离、富集蔬菜中病毒粒子能够提高检测的准确度和灵敏度。该文采用甘氨酸缓冲液洗脱-PEG 6000沉淀富集蔬菜中的诺如病毒和轮状病毒，对影响分离、富集效果的主要因素(甘氨酸缓冲液pH，PEG 6000的浓度和沉淀时间)进行优化，最后采用RT-qPCR检测病毒。结果表明：采用pH5.0的甘氨酸缓冲液洗脱，15% PEG 6000沉淀洗脱液中的病毒粒子2 h，样品中病毒回收率最大，诺如病毒回收率为13.35%~20.30%，轮状病毒回收率为18.08%~20.00%。该研究可为蔬菜中病毒的准确检测提供方法学参考。
Study on concentration of foodborne viruses in vegetables
RUAN Jia1,2, ZHANG Weiwei1, LIU Xin1, SU Huilan1
1. Chengdu Medical College, Chengdu 621000, China;
2. West China School of Public Health, Sichuan Univercity, Chengdu 610041, China
Abstract: Vegetables are in a high risk of food poisoning after contaminated with norovirus and rotavirus. It’s critical to detect the two foodborne viruses accurately in vegetables for ensuring food safety. To separate and concentrate the viral particles in vegetables can improve the detection accuracy and sensitivity obviously. In this research, glycine buffer was used for eluting the viral particles from vegetables. Subsequently, the separated viral particles were concentrated by PEG 6000 precipitation. The main factors (the pH of the glycine buffer, the concentration and the precipitation time of the PEG 6000) affecting the recovery rate of the pretreatment method were optimized. The concentrated viral particles were detected by RT-qPCR. When the pH of glycine buffer were 5.0, and PEG 6000 concentration were 15% and precipitation time were 2 hours, the optimal recovery rate was obtained. The recovery rates of norovirus and rotavirus were 13.35%-20.30% and 18.08%-20.00%, respectively. This research provides a methodological reference for the accurate detection of virus in vegetables.
Keywords: norovirus;rotavirus;lettuce;concentration method
2020, 46(10):11-17 收稿日期: 2020-07-10;收到修改稿日期: 2020-08-12
基金项目: 国家自然科学基金资助项目（81401757）；四川省科技计划项目应用基础研究（2019YJ0367）；四川省科技创新苗子工程资助项目（2019047）；成都医学院科研基金自科项目（17Z137）
作者简介: 阮佳(1990-),女,四川三台县人,讲师,博士,主要从事卫生检验与检疫工作
参考文献
[1] WANG J, ZHOU H, MO Z, et al. Burden of viral gastroenteritis in children living in rural China: Population-based surveillance[J]. International Journal of Infectious Diseases, 2020, 90: 151-160
[2] LI B, XIAO D, LI Y, et al. Epidemiological analysis of norovirus infectious diarrhea outbreaks in Chongqing, China, from 2011 to 2016[J]. Journal of Infection and Public Health, 2020, 13(1): 46-50
[3] 佟瑞, 楼永良, 谭文杰. 新近发现的细小病毒与多瘤病毒感染[J]. 中华实验和临床病毒学杂志, 2011(5): 396-398
[4] DESSELBERGER U, GRAY J. Gastrointestinal infections viral gastroenteritis[J]. Ulrich Desselberger, Medicine, 2017, 45(11): 690-694
[5] BANYAI K, ESTES M, MARTELLA V, et al. Viral gastroenteritis[J]. The Lancet, 2018, 392(10142): 175-186
[6] ANSARI S A, SUSAN S V, SATTAR S A. Survival and vehicular spread of human rotaviruses: possible relation to seasonality of outbreaks[J]. Reviews of Infectious Diseases, 1991, 13(3): 448-461
[7] CANNON, JENNIFER L. Surrogates for the study of norovirus stability and inactivation in the environment: a comparison of murine norovirus and feline calicivirus.[J]. Journal of Food Protection, 2006, 69(11): 2761-2765
[8] SCHERER K, JOHNE R, SCHRADER C, et al. Comparison of two extraction methods for viruses in food and application in a norovirus gastroenteritis outbreak[J]. Journal of Virological Methods, 2010, 169(1): 22-27
[9] HENNECHART-COLLETTE C, NIVEAU F, MARTIN-LATIL S, et al. Development of an extraction method to detect enteric viruses in dressed vegetables[J]. International Journal of Food Microbiology, 2019, 311(2): 108349
[10] WU R, XING X, CORREDIG M, et al. Concentration of hepatitis A virus in milk using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles[J]. Food Microbiology, 2019, 84: 103236
[11] GOYAL S M, ABOUBAKR H A. Methods for virus recovery from foods[M]//Viruses in Foods, Food Microbiology and Food Safety. Switzerland: Springer International Publishing, 2016: 231-276.
[12] PURPARI G, MACALUSO G, Di BELLA S, et al. Molecular characterization of human enteric viruses in food, water samples, and surface swabs in Sicily[J]. International Journal of Infectious Diseases, 2019, 80: 66-72
[13] PANG X L, PREIKSAITIS J K, LEE B. Multiplex real time RT-PCR for the detection and quantitation of norovirus genogroups I and II in patients with acute gastroenteritis[J]. Journal of Clinical Virology, 2005, 33(2): 168-171
[14] ZENG S Q, HALKOSALO A, SALMINEN M, et al. One-step quantitative RT-PCR for the detection of rotavirus in acute gastroenteritis[J]. Journal of Virological Methods, 2008, 153(2): 238-240
[15] RZEZUTKA A, ALOTAIBI M, D'AGOSTINO M, et al. A centrifugation-based method for extraction of norovirus from raspberries[J]. Journal of Food Protection, 2005, 68(9): 1923-1925
[16] LE G F, SCHULTZ A C, HAUGARREAU L, et al. Round-robin comparison of methods for the detection of human enteric viruses in lettuce[J]. Journal of Food Protection, 2004, 67(10): 2315-2319
[17] CROCI L, De MEDICI D, SCALFARO C, et al. The survival of hepatitis A virus in fresh produce[J]. International Journal of Food Microbiology, 2002, 73(1): 29-34
[18] COUDRAY C, MERLE G, MARTIN-LATIL S, et al. Comparison of two extraction methods for the detection of hepatitis A virus in lettuces using the murine norovirus as a process control[J]. Journal of Virological Methods, 2013, 193(1): 96-102
[19] CROCI L, DUBOIS E, COOK N, et al. Current methods for extraction and concentration of enteric viruses from fresh fruit and vegetables: towards international standards[J]. Food Anal. Methods, 2008, 1(2): 73-84
[20] 苏来金, 柳淑芳, 李振, 等. 水产品中诺如病毒富集方法的优化研究[J]. 渔业科学进展, 2008, 29(6): 126-132
[21] AUSAR S F, FOUBERT T R, HUDSON M H, et al. Conformational stability and disassembly of norwalk virus-like particles: effect of pH and temperature[J]. Journal of Biological Chemistry, 2006, 281(28): 19478-19488
[22] WHITE L J, HARDY M E, ESTES M K. Biochemical characterization of a smaller form of recombinant Norwalk virus capsids assembled in insect cells[J]. Journal of Virology, 1997, 71(10): 8066-8072
[23] 殷绥亚. 聚乙二醇用于病毒和噬菌体的浓缩与提纯[J]. 国际生物制品学杂志, 1979(1): 21-23
[24] 李巧艳, 吴新伟, 刘于飞, 等. 利用荧光RT-PCR法检测环境水体中诺如病毒[J]. 中国卫生检验杂志, 2011(2): 132-135
[25] LEWIS G D, METCALF T G. Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples[J]. Applied & Environmental Microbiology, 1988, 54(8): 1983-1988
[26] 陈莉萍, 徐德顺. 水中病毒浓缩方法研究进展[J]. 中国卫生检验杂志, 2015, 25(2): 301-304
[27] PENG T, ANNA E, ROBERT M. Two-log increase in sensitivity for detection of norovirus in complex samples by concentration with porcine gastric mucin conjugated to magnetic beads.[J]. Applied & Environmental Microbiology, 2008, 74(14): 4271-4276
[28] 林虹, 管大伟, 邓小玲, 等. 应用TaqMan荧光定量RT-PCR检测猪肝中戊型肝炎病毒的研究[J]. 华南预防医学, 2015, 41(2): 171-175
[29] 王晓辉, 龙润乡, 张名, 等. HAV病毒液的3种浓缩方法比较[J]. 现代生物医学进展, 2014, 14(24): 4616-4619
