食源性诺如病毒在果蔬农产品中的污染及检测研究

The Advances in the Contamination and Detection of Foodborne Pathogen Noroviruses in Fresh Produce

本文综述了食源性致病微生物-诺如病毒(Norovirus,NoV)在果蔬农产品中的来源和分布、在果蔬农产品中的富集情况、与果蔬农产品相关的食源性NoV暴发情况以及在农产品中的检测方法等内容。近30年来,与农产品相关的食源性NoV暴发数目持续增长。这主要与NoV的传播特性(强传染性、低感染剂量和对环境的较强抗性)以及近15年食品中NoV检测方法的显著发展有关。生鲜农产品作为高风险的食品,通常不经烹饪直接生食,在供应链中不同环节都容易被污染,如人类粪便污染的灌溉用水、肥料、食物处理用水、食物处理者的不卫生操作、交叉污染等。NoV在果蔬农产品中的结合主要与病毒颗粒的理化性质及农产品对NoV的吸附和可能的内在化途径有关。根据欧美国家与果蔬农产品有关的食源性NoV暴发的最新数据统计分析,在欧洲大规模食源性暴发主要由被NoV污染的覆盆子和生菜导致,在美国主要由被NoV污染的生菜导致。在国内,由于尚无农产品中食源性NoV检测的相关研究,缺乏与农产品相关NoV暴发的相关数据,因此无法对国内农产品中NoV的污染情况进行具体分析。农产品中NoV具有含量低且分布不均匀的特点,针对不同样品,需要采用合理的采样方法、病毒核糖核酸(Ribonucleic acid,RNA)提取方法以及实时荧光定量PCR(Real-time quantitative polymerase chain reaction,RT-qPCR)方法对其进行检测。根据NoV外壳蛋白的物化特性,高pH值和高离子强度的洗脱溶液(如碱性甘氨酸缓冲液等)可提高NoV的回收率;对于酸性水果中的病毒洗脱,加入三羟甲基氨基甲烷-盐酸(Tris(hydroxymethyl)aminomethane-HCl,Tris-HCl)可以提高回收率;莓果类中含有的果胶,可在中性条件下用果胶酶去除。本文还论述了食品中食源性NoV检测的国际标准ISO/TS 15216:2013、果蔬农产品中NoV检测方法的汇总以及检测NoV的最新方法(原位杂交RT-qPCR方法),为开展农产品中NoV检测的研究提供部分参考,从而建立和发展农产品中NoV的检测方法,针对不同果蔬产品对NoV的吸附特性,配套相应NoV的提取方法,建立多种农产品中NoV常规分子检测方法,以利于对农产品安全的源头监控。

This article reviewed the researches proceeding on the contamination and detection of the foodborne pathogen noroviruses（NoVs）in fresh produce,which involved the NoVs contaminations in fresh produce,the special attachment of NoVs in fresh produce,the NoVs outbreaks associated with fresh produce and the NoVs detection in fresh produce.There had been an increase in reported infectious disease risks associated with the consumptions of fresh produce for recent 30 years.Because the NoVs,as a primary cause of viral gastroenteritis thoughout the world,were highly contagious,had a low infectious dose,and were persistent in the environment.And also the methods for NoVs detection in food had significantly developed over the last 15 years.Currently NoVs were the most common pathogen accounting for 40% of outbreaks associated with fresh produce（i.e.,fruits and vegetables）.Data from outbreaks investigations verified fresh produce as the high risk food products for NoVs.The fresh produce were typically eaten raw with no thermal processing,can be contaminated at any step during production and processing from faecally polluted water and fertilizers,the poor hygiene practices by food handlers and the cross-contamination.The attachment of NoVs to the fresh produce was due to the physio-chemical factors of virus protein coat,the special attachment to different fresh produce,and the possibility for internalization of NoVs.It might provide answers to why those high risk foods were more frequently implicated（i.e.,lettuce and raspberries）.According to the data of foodborne NoVs outbreaks which were associated with fresh produce from EU countries and the USA,the outbreaks in EU countries were mainly associated with NoVs contaminated raspberries and lettuce,while in USA which were associated with NoVs contaminated lettuce.Unfortunately,there were no NoVs detection methods for fresh produce or the data of foodborne NoVs outbreaks which were associated with fresh produce in China.That made it difficult to analyze the NoVs contamination situation in China.The heterogeneous distributions of presumably low levels of virus,which presented in contaminated fresh produce,also made it difficult to detect NoVs.To solve this problem,different sampling methods,viral elution methods and RT-qPCR methods were chosen.For example,according to the isoelectric point of NoVs particles,high pH and high ionic strength solution could be used as means for releasing NoVs.For the elution from acidic fruit,the buffer capacity and the virus recovery could be increased by the addition of tris-HCl.When analyzing pectin containing raspberries or strawberries,the viral elution usually incubated with pectinase at neutral pH to avoid from foaming jelly.In this paper,the latest ISO standard for NoV detection in food and the new approaches for NoV detection were also reviewed to provide references for domestic researches.It was necessary to establish and develop domestic methods for NoV detection in fresh produce,especially the different NoV conventional molecular detection methods with corresponding NoV extraction methods,which targeted to the different adsorption characteristics of different fruits and vegetables,in order to strengthen the national food safety monitoring.