不同食物中诺如病毒检测方法的优化与评估
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摘要
近年来,由病毒引起的食源性疾病越来越多,其中诺如病毒(Noroviruses,NoVs)是全球急性胃肠炎暴发的主要致病原。污染的食品是NoVs主要的传播途径之一。本文对不同食品中NoVs检测方法进行优化和评估,为我国NoVs引起的食源性病毒病的应对提供实验室检测技术支撑。以鼠诺如病毒(Murine norovirus-1,MNV-1)为模式病毒和过程对照病毒,优化和评估洗脱-浓缩法和直接RNA提取法,考察Trallk洗液、NaPP洗液、TGBE洗液和TPB洗液对新鲜果蔬食品(包括草莓、葡萄、洋葱和生菜)中MNV-1的洗脱效果,以及不同浓缩方法(PEG沉淀法和超滤法)对病毒洗脱液中MNV-1的浓缩效果;比较洗脱-浓缩法和直接RNA提取法对蛋白、脂肪类食品(蛋糕和午餐肉)中MNV-1的检测效果;评估优化后的方法对新鲜果蔬食品和蛋白、脂肪类食品中NoVs GII的检测效果。结果表明草莓、葡萄、生菜、洋葱中MNV-1的最优洗脱-浓缩方法分别是TGBE洗液-PEG沉淀组合、TGBE洗液-超滤组合、Trallk洗液-PEG沉淀组合和Trallk洗液-超滤组合。草莓和生菜中NoVs GII的检测限可达103基因拷贝数/10g,而葡萄和洋葱中NoVs GII的检测限为10~4基因拷贝数/10g。洗脱-浓缩法和直接RNA提取法回收蛋糕和午餐肉中MNV-1后的效果显示,前者没有回收到MNV-1,后者却成功回收到MNV-1,但是NoVs GII的检测限高于其他种类食品,分别为10~6基因拷贝数/2g和10~4基因拷贝数/2g。本研究建立了新鲜果蔬食品和蛋白脂肪类食品中NoVs GII的检测方法,从而优化和完善了我国食品中NoVs检测体系。
In recent years,there have been more and more food-borne diseases caused by viruses. Among them,Noroviruses(NoVs)are the main cause of acute gastroenteritis in the world. Contaminated food is one of the main transmission route of NoVs. The present study aims to optimizing and evaluating the detection methods of NoVs in different foods,and providing laboratory technologies for the surveillance of food-borne viral diseases in China. The elution-concentration methods and direct RNA extraction method were optimized and evaluated using Murine Norovirus-1(MNV-1)as a model virus and a process control virus. The effects of Trallk lotion,NaPP lotion,TGBE lotion and TPB lotion on MNV-1 in fresh fruits and vegetables(including strawberries,grapes, onions, and lettuces), and the concentration effect of different concentration methods(PEG precipitation method and ultrafiltration method)on MNV-1 in the virus eluation were investigated. The effects of elution-concentration and direct RNA extraction on MNV-1 in protein and fat foods(cake and luncheon meat)were compared. The effect of the optimized methods on the detection of NoVs in fresh fruits,vegetables,protein and fat foods were evaluated. The results showed that the optimal elution and concentration methods of MNV-1 in strawberries,grapes,lettuces,and onions were the combination of TGBE lotion-PEG precipitation,TGBE lotion-ultrafiltration,Trallk lotion-PEG precipitation and Trallk lotion-ultrafiltration. The detection limit for NoVs is 103 genecopies/10 g in strawberries and lettuces,while the detection limit is 10~4 genecopies/10 g in grapes and onions. The recovery of MNV-1 in cakes and luncheon meat by the elution-concentration methods and direct RNA extraction showed that the former did not detect MNV-1 from cakes and luncheon meat,however the latter successfully recovered MNV-1. The detection limit of NoVs was 10~6 genecopies/2 g and 10~4 genecopies/2 g for cakes and luncheon meat respectively,higher than other types of food. This study established the methods for the detection of NoVs in fresh fruits and vegetables as well as protein and fat foods,and optimized and perfected the NoVs detection system of food in China.
In recent years,there have been more and more food-borne diseases caused by viruses. Among them,Noroviruses(NoVs)are the main cause of acute gastroenteritis in the world. Contaminated food is one of the main transmission route of NoVs. The present study aims to optimizing and evaluating the detection methods of NoVs in different foods,and providing laboratory technologies for the surveillance of food-borne viral diseases in China. The elution-concentration methods and direct RNA extraction method were optimized and evaluated using Murine Norovirus-1(MNV-1)as a model virus and a process control virus. The effects of Trallk lotion,NaPP lotion,TGBE lotion and TPB lotion on MNV-1 in fresh fruits and vegetables(including strawberries,grapes, onions, and lettuces), and the concentration effect of different concentration methods(PEG precipitation method and ultrafiltration method)on MNV-1 in the virus eluation were investigated. The effects of elution-concentration and direct RNA extraction on MNV-1 in protein and fat foods(cake and luncheon meat)were compared. The effect of the optimized methods on the detection of NoVs in fresh fruits,vegetables,protein and fat foods were evaluated. The results showed that the optimal elution and concentration methods of MNV-1 in strawberries,grapes,lettuces,and onions were the combination of TGBE lotion-PEG precipitation,TGBE lotion-ultrafiltration,Trallk lotion-PEG precipitation and Trallk lotion-ultrafiltration. The detection limit for NoVs is 103 genecopies/10 g in strawberries and lettuces,while the detection limit is 10~4 genecopies/10 g in grapes and onions. The recovery of MNV-1 in cakes and luncheon meat by the elution-concentration methods and direct RNA extraction showed that the former did not detect MNV-1 from cakes and luncheon meat,however the latter successfully recovered MNV-1. The detection limit of NoVs was 10~6 genecopies/2 g and 10~4 genecopies/2 g for cakes and luncheon meat respectively,higher than other types of food. This study established the methods for the detection of NoVs in fresh fruits and vegetables as well as protein and fat foods,and optimized and perfected the NoVs detection system of food in China.
引文
[1]刘江艺,黄永翰,李锋平,吕文辉.国内诺如病毒性胃肠炎暴发疫情影响因素的Meta分析[J].公共卫生与预防医学,2017,28(6):79-82.
[2]Verhoef L,Hewitt J,Barclay L,Ahmed S M,Lake R,Hall A J,Lopman B,Kroneman A,Vennema H,Vinje J,Koopmans M.Norovirus genotype profiles associated with foodborne transmission,1999-2012[J].Emerg In-fect Dis,2015,21(4):592-599.
[3]谢雅晶,刘贤金.食源性诺如病毒在果蔬农产品中的污染及检测研究[J].病毒学报,2015,31(6):685-697.DOI:10.13242/j.cnki.bingduxuebao.002831
[4]周冬梅,靳淼,李慧莹,徐子乾,段招军.双重荧光定量一步RT-PCR法同时检测GI、GⅡ型诺如病毒[J].病毒学报,2013,29(3):310-315.DOI:10.13242/j.cnki.bingduxuebao.002392
[5]Iso.Microbiology of food and animal feed horizontal method for determination of hepatitis A virus and norovi-rus in food using real-time RT-PCR,in Part 2:Method for qualitative detection[S].ISO/TS 15216-2 ed.2013.
[6]Wobus C E,Thackray L B,Virgin H T.Murine norovi-rus:a model system to study norovirus biology and pathogenesis[J].J Virol,2006,80(11):5104-5112.
[7]Hennechart-Collette C,Martin-Latil S,Guillier L,Per-elle S.Determination of which virus to use as a process control when testing for the presence of hepatitis A virus and norovirus in food and water[J].Int J Food Microbi-ol,2015,202:57-65.
[8]李晖,黄吉成,方苓,严纪文,邹丽容,陈秋霞,黄平.贝类海产品中诺如病毒快速检测方法研究[J].中国卫生检验杂志,2006,16(11):1300-1302.
[9]吴斌,裴轶君,王刚,李叶.贝类中诺如病毒ELISA与荧光定量PCR检测技术的研究[J].中国卫生检验杂志,2008,18(12):2623-2624.
[10]Praveen C,Dancho B A,Kingsley D H,Calci K R,Meade G K,Mena K D,Pillai S D.Susceptibility of mu-rine norovirus and hepatitis A virus to electron beam irra-diation in oysters and quantifying the reduction in poten-tial infection risks[J].Appl Environ Microbiol,2013,79(12):3796-3801.
[11]Kim H Y,Kwak I S,Hwang I G,Ko G.Optimization of methods for detecting norovirus on various fruit[J].JVirol Methods,2008,153(2):104-110.
[12]Cheong S,Lee C,Choi W C,Lee C H,Kim S J.Con-centration method for the detection of enteric viruses from large volumes of foods[J].J Food Prot,2009,72(9):2001-2005.
[13]Herrmann J E,Cliver D O.Food-borne virus:detection in a model system[J].Appl Microbiol,1968,16(4):595-602.
[14]Baert L,Uyttendaele M,Debevere J.Evaluation of vi-ral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Noro-virus GII detection[J].Int J Food Microbiol,2008,123(1-2):101-108.
[15]Morton V,Jean J,Farber J,Mattison K.Detection of noroviruses in ready-to-eat foods by using carbohydratecoated magnetic beads[J].Appl Environ Microbiol,2009,75(13):4641-4643.
[16]张其刚,潘良文,李想,方筠.猪胃黏蛋白偶联磁珠和聚乙二醇富集检测青葱和葡萄中诺如病毒的比较研究[J].食品科学,2012,33(16):241-245.
[17]周晓红.食品与水中诺如病毒检测方法的建立及其在疫情暴发中的初步应用[D].暨南大学,2010.
[18]管锦绣,许喜林,翁文川,凌莉,刘静宇,冼钰茵.西生菜中低污染量GⅡ型诺如病毒的富集与定量检测研究[J].食品安全质量检测学报,2017,8(9):3536-3542.
[19]Stals A,Baert L,De K A,Van C E,Uyttendaele M.Evaluation of a norovirus detection methodology for ready-to-eat foods[J].Int J Food Microbiol,2011,145(2-3):420-425.
[2]Verhoef L,Hewitt J,Barclay L,Ahmed S M,Lake R,Hall A J,Lopman B,Kroneman A,Vennema H,Vinje J,Koopmans M.Norovirus genotype profiles associated with foodborne transmission,1999-2012[J].Emerg In-fect Dis,2015,21(4):592-599.
[3]谢雅晶,刘贤金.食源性诺如病毒在果蔬农产品中的污染及检测研究[J].病毒学报,2015,31(6):685-697.DOI:10.13242/j.cnki.bingduxuebao.002831
[4]周冬梅,靳淼,李慧莹,徐子乾,段招军.双重荧光定量一步RT-PCR法同时检测GI、GⅡ型诺如病毒[J].病毒学报,2013,29(3):310-315.DOI:10.13242/j.cnki.bingduxuebao.002392
[5]Iso.Microbiology of food and animal feed horizontal method for determination of hepatitis A virus and norovi-rus in food using real-time RT-PCR,in Part 2:Method for qualitative detection[S].ISO/TS 15216-2 ed.2013.
[6]Wobus C E,Thackray L B,Virgin H T.Murine norovi-rus:a model system to study norovirus biology and pathogenesis[J].J Virol,2006,80(11):5104-5112.
[7]Hennechart-Collette C,Martin-Latil S,Guillier L,Per-elle S.Determination of which virus to use as a process control when testing for the presence of hepatitis A virus and norovirus in food and water[J].Int J Food Microbi-ol,2015,202:57-65.
[8]李晖,黄吉成,方苓,严纪文,邹丽容,陈秋霞,黄平.贝类海产品中诺如病毒快速检测方法研究[J].中国卫生检验杂志,2006,16(11):1300-1302.
[9]吴斌,裴轶君,王刚,李叶.贝类中诺如病毒ELISA与荧光定量PCR检测技术的研究[J].中国卫生检验杂志,2008,18(12):2623-2624.
[10]Praveen C,Dancho B A,Kingsley D H,Calci K R,Meade G K,Mena K D,Pillai S D.Susceptibility of mu-rine norovirus and hepatitis A virus to electron beam irra-diation in oysters and quantifying the reduction in poten-tial infection risks[J].Appl Environ Microbiol,2013,79(12):3796-3801.
[11]Kim H Y,Kwak I S,Hwang I G,Ko G.Optimization of methods for detecting norovirus on various fruit[J].JVirol Methods,2008,153(2):104-110.
[12]Cheong S,Lee C,Choi W C,Lee C H,Kim S J.Con-centration method for the detection of enteric viruses from large volumes of foods[J].J Food Prot,2009,72(9):2001-2005.
[13]Herrmann J E,Cliver D O.Food-borne virus:detection in a model system[J].Appl Microbiol,1968,16(4):595-602.
[14]Baert L,Uyttendaele M,Debevere J.Evaluation of vi-ral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Noro-virus GII detection[J].Int J Food Microbiol,2008,123(1-2):101-108.
[15]Morton V,Jean J,Farber J,Mattison K.Detection of noroviruses in ready-to-eat foods by using carbohydratecoated magnetic beads[J].Appl Environ Microbiol,2009,75(13):4641-4643.
[16]张其刚,潘良文,李想,方筠.猪胃黏蛋白偶联磁珠和聚乙二醇富集检测青葱和葡萄中诺如病毒的比较研究[J].食品科学,2012,33(16):241-245.
[17]周晓红.食品与水中诺如病毒检测方法的建立及其在疫情暴发中的初步应用[D].暨南大学,2010.
[18]管锦绣,许喜林,翁文川,凌莉,刘静宇,冼钰茵.西生菜中低污染量GⅡ型诺如病毒的富集与定量检测研究[J].食品安全质量检测学报,2017,8(9):3536-3542.
[19]Stals A,Baert L,De K A,Van C E,Uyttendaele M.Evaluation of a norovirus detection methodology for ready-to-eat foods[J].Int J Food Microbiol,2011,145(2-3):420-425.