含4 种食源性病毒检测靶标多联装甲RNA的制备、纯化与定值

基于Qβ噬菌体装甲RNA制备平台构建同时含有诺如病毒（norovirus,NoV）、甲肝病毒（hepatitis A virus,HAV）、轮状病毒（rotavirus,RV）、星状病毒（astrovirus,AstV）检测靶标RNA的多联装甲RNA（multiplex armored RNA,AR-MulV）,并进行纯化与初步定值。结果表明,重组质粒在大肠杆菌中成功表达,表达产物大小约为14.1 kDa;制备的AR-MulV经纯化后无杂蛋白与残留重组质粒,电镜下可见大量结构形态完整的病毒样颗粒,大小约为25 nm。初步定值结果显示,AR-MulV中GI型NoV、GII型NoV、HAV、RV和AstV检测靶标RNA的含量分别为（1.24±0.2）×107、（2.54±0.6）×107、（2.24±0.3）×107、（2.96±0.5）×107 copies/μL和（3.19±0.4）×107 copies/μL。本研究基于Qβ噬菌体装甲RNA制备平台成功制备同时包含4 种食源性病毒标准方法检测靶标的多联装甲RNA,为食源性病毒的分子检测以及多重实时荧光定量逆转录-聚合酶链式反应阳性质控样品的研发提供新思路。     

果蔬食品中诺如病毒污染状况及检测研究进展

食源性疾病不但严重危害人们的健康, 而且造成大量的经济损失。在全球范围内与果蔬等农产品相关的诺如病毒(Norovirus, NoV)污染事件持续暴发并呈增长态势。由于果蔬类食品中诺如病毒污染含量较低, 再加上食品基质成分的干扰, 使得果蔬制品中的NoV检测成为一项非常艰巨的任务。传统的检测技术面临许多缺点, 例如鉴定的特异性有待增强; 提取效率不够高, 检测敏感性尚需改进。这意味着需要转换视角, 开展食源性诺如病毒检测技术的创新研究。本文综述了果蔬食品中NoV污染流行状况及易受污染的、需加以关注的果蔬食品种类, 总结了果蔬食品中NoV洗脱及富集方法、介绍了基于组学的检测技术及发展趋势, 希望为不断提高果蔬食品中NoV检测防控能力提供参考。     

蓝莓中诺如病毒检测方法的优化及其在果蔬中的应用

目的:建立蓝莓等浆果类和蔬菜类食品中GII型诺如病毒(NoV GII)的有效检测方法。方法:利用已知NoV GII病毒样本对阴性的蓝莓进行人工染毒,分别比较7种洗脱液和2种浓缩方法的回收效果,优化RNA提取方法,最后采用荧光定量PCR方法进行检测。用新建立的诺如病毒浓缩检测方法,对7种市购浆果和蔬菜样品进行检测并研究其最低检出限。结果:实时荧光RT-PCR检测结果表明,人工染毒后的蓝莓等浆果样品(表皮和基质)采用TP ALK洗脱液洗脱,经酶处理后超滤法浓缩,最后采用病毒核酸提取试剂盒(QIAamp Viral RNA Mini Kit)提取浓缩病毒的RNA,诺如病毒的回收效率达到较高的水平,其灵敏度达3.5～350 PCRU/10g。将该方法用于草莓、杨梅、葡萄、小番茄、生菜、胡萝卜、黄瓜等7种水果蔬菜中诺如病毒的检测,其中杨梅和小番茄的检测灵敏度达到3.5～3 500 PCRU/10 g,草莓、生菜和胡萝卜次之,为35～35 000 PCRU/10 g,黄瓜和葡萄的灵敏度较低。结论:所建立的果蔬中诺如病毒富集方法和核酸提取方法操作简便,灵敏度较高,适合于蓝莓等浆果和蔬菜类样品中NoV GII的检测。     

舟山市海产贝类和食源性腹泻病例中肠道腹泻病毒监测

目的了解肠道腹泻病毒在舟山市海产贝类的分布和食源性腹泻病例中的感染特征以及两者相关联系,为预防和控制食源性疾病提供有效的对策和措施。方法采用荧光定量聚合酶链式反应(PCR)方法对466份舟山市海产贝类和422份食源性腹泻标本中诺如病毒(NoV)GⅠ型/GⅡ型、轮状病毒(RV)、札如病毒(SPV)、星状病毒(AsV)和肠道腺病毒(AdV)5种主要肠道腹泻病毒核酸进行检测。结果 466份海产贝类样品病毒总阳性率为18.03%(84/466),其中NoV GⅡ型为4.08%(19/466),SPV为9.67%(45/466),AsV为2.79%(13/466),AdV为1.50%(7/466);春季和冬季病毒阳性率较高,农贸市场的牡蛎病毒阳性率最高(24.75%,25/101),养殖毛蚶病毒阳性率最低(10.12%,17/168),不同的病毒阳性率以及不同来源和不同季节贝类的病毒阳性率差异均有统计学意义(P0.05)。422例腹泻患者粪便标本总阳性率为15.64%(66/422),NoV为4.74%(20/422,以GⅡ为主),RV为4.74%(20/422),SPV为3.55%(15/422),NoV GⅡ型、RV和SPV的阳性率相对较高,春季和冬季病毒阳性率较高,不同病毒阳性率和不同季节病毒阳性率差异有统计学意义(P0.05)。结论舟山市海产贝类中肠道腹泻病毒的污染状况较为严重,与食源性腹泻病例中的病毒感染特征存在相关联系,其流行季节和优势病毒类型相同。     

GI和GII诺如病毒RAA可视化快速检测方法的建立

诺如病毒(NoV)是引起全球急性肠胃炎疾病的主要病原体之一，极易爆发传播，增加医疗与经济负担。目的:通过重组酶介导等温扩增技术(RAA)开发一种新型的NoV检测方法。方法:根据ISO TS 15216-2-2013、GB 4789.42规定的GI和GII NoV检测靶标所对应的cDNA序列，设计并筛选最佳RAA引物及探针，确定其对其它常见食源性腹泻病毒的特异性。通过优化确定最短检测时间、反应程序以及反应体系，分析该检测体系下对NoV参考质粒和真实样本检测的灵敏度，从而建立GI和GII NoV RAA可视化快速检测方法。结果:所建立的RAA检测方法特异性好，与其它食源性病毒无交叉反应。在保证扩增效率的前提下，优化后的反应程序可将检测时间缩短为10 min左右，反应成本可减少三分之二。对参考质粒和真实样本检测的灵敏度分别达10-2 ng/μL和1 ng/μL。结论:本试验建立的两种NoV检测方法特异性强、灵敏度高，且简单、快速、可视，为今后NoV快速检测奠定良好的基础。     

不同水源中GⅡ型诺如病毒实时荧光逆转录聚合酶链式反应检测方法建立及应用

目的建立不同水源中GⅡ型诺如病毒(NoV GⅡ)实时荧光逆转录聚合酶链式反应(RT-PCR)检测方法 ,对某市疾病预防控制中心送检的10份疑似引起诺如病毒中毒的水样进行NoV GⅡ检测。方法以瓶装水、河水和生活污水为研究对象,采用硝酸纤维素膜-聚乙二醇(PEG)沉淀法富集病毒,对取样体积、病毒洗脱条件、PEG终浓度及PEG沉淀条件等进行了优化,提取病毒RNA、建立实时荧光RT-PCR检测方法 ;通过外加MS2计算方法的回收率,评价所建方法对水样中诺如病毒的回收效果。结果建立的方法对瓶装水、河水和生活污水中NoV GⅡ的平均回收率分别为(60.1±8.0)%、(22.0±6.5)%和(35.7±8.1)%,10份送检水样中3份检出NoV GⅡ。结论建立的实时荧光RT-PCR方法适用于瓶装水、河水和生活污水中NoV GⅡ的检测,饮用水被NoV GⅡ是引发某市人员中毒的原因之一。     

食源性甲型肝炎病毒快速检测技术研究进展

病毒在食源性疾病的爆发中起着至关重要的作用,其中,甲型肝炎病毒(hepatitis A virus,HAV)感染后能够致人衰弱,并引发急性肝衰竭。由于其传染迅速,影响范围广, HAV污染已经给消费者带来了严重的健康风险,且在全球范围内造成了大规模的食源性疫情和经济损失。因此,早期快速准确的检测HAV对于食品安全和疫情暴发的溯源至关重要,以便及时确定并召回受污染的食品,防止感染的进一步发生。传统的HAV检测方法由于检出率不高以及病毒在细胞内的培养周期长等问题,往往耗时费力,无法快速有效地对其进行检测。本文对目前报道较多的HAV快速检测技术进行了阐述和梳理,对各项技术的检出限、检测时间以及优缺点进行了比较,并对HAV快速检测技术研究进行了展望,为后续HAV的快速检测研究提供依据,也有助于进一步探讨与开发HAV快速检测的新技术,以有效防止HAV的传播与危害。     

Foodborne viruses: an emerging problem

Several groups of viruses may infect persons after ingestion and then are shed via stool. Of these, the norovirus (NoV) and hepatitis A virus (HAV) are currently recognised as the most important human foodborne pathogens with regard to the number of outbreaks and people affected in the Western world. NoV and HAV are highly infectious and may lead to widespread outbreaks. The clinical manifestation of NoV infection, however, is relatively mild. Asymptomatic infections are common and may contribute to the spread of the infection. Introduction of NoV in a community or population (a seeding event) may be followed by additional spread because of the highly infectious nature of NoV, resulting in a great number of secondary infections (50% of contacts). Hepatitis A is an increasing problem because of the decrease in immunity of populations in countries with high standards of hygiene. Molecular-based methods can detect viruses in shellfish but are not yet available for other foods. The applicability of the methods currently available for monitoring foods for viral contamination is unknown. No consistent correlation has been found between the presence of indicator microorganisms (i.e. bacteriophages, E. coli) and viruses. NoV and HAV are highly infectious and exhibit variable levels of resistance to heat and disinfection agents. However, they are both inactivated at 100 degrees C. No validated model virus or model system is available for studies of inactivation of NoV, although investigations could make use of structurally similar viruses (i.e. canine and feline caliciviruses). In the absence of a model virus or model system, food safety guidelines need to be based on studies that have been performed with the most resistant enteric RNA viruses (i.e. HAV, for which a model system does exist) and also with bacteriophages (for water). Most documented foodborne viral outbreaks can be traced to food that has been manually handled by an infected foodhandler, rather than to industrially processed foods. The viral contamination of food can occur anywhere in the process from farm to fork, but most foodborne viral infections can be traced back to infected persons who handle food that is not heated or otherwise treated afterwards. Therefore, emphasis should be on stringent personal hygiene during preparation. If viruses are present in food preprocessing, residual viral infectivity may be present after some industrial processes. Therefore, it is key that sufficient attention be given to good agriculture practice (GAP) and good manufacturing practice (GMP) to avoid introduction of viruses onto the raw material and into the food-manufacturing environment, and to HACCP to assure adequate management of (control over) viruses present during the manufacturing process. If viruses are present in foods after processing, they remain infectious in most circumstances and in most foods for several days or weeks, especially if kept cooled (at 4 degrees C). Therefore, emphasis should be on stringent personal hygiene during preparation. For the control of foodborne viral infections, it is necessary to: Heighten awareness about the presence and spread of these viruses by foodhandlers; Optimise and standardise methods for the detection of foodborne viruses; Develop laboratory-based surveillance to detect large, common-source outbreaks at an early stage; and Emphasise consideration of viruses in setting up food safety quality control and management systems (GHP, GMP, HACCP).     

Analytical Methods for Virus Detection in Water and Food

Potential ways to address the issues that relate to the techniques for analyzing food and environmental samples for the presence of enteric viruses are discussed. It is not the authors?? remit to produce or recommend standard or reference methods but to address specific issues in the analytical procedures. Foods of primary importance are bivalve molluscs, particularly, oysters, clams, and mussels; salad crops such as lettuce, green onions and other greens; and soft fruits such as raspberries and strawberries. All types of water, not only drinking water but also recreational water (fresh, marine, and swimming pool), river water (irrigation water), raw and treated sewage are potential vehicles for virus transmission. Well over 100 different enteric viruses could be food or water contaminants; however, with few exceptions, most well-characterized foodborne or waterborne viral outbreaks are restricted to hepatitis A virus (HAV) and calicivirus, essentially norovirus (NoV). Target viruses for analytical methods include, in addition to NoV and HAV, hepatitis E virus (HEV), enteroviruses (e.g., poliovirus), adenovirus, rotavirus, astrovirus, and any other relevant virus likely to be transmitted by food or water. A survey of the currently available methods for detection of viruses in food and environmental matrices was conducted, gathering information on protocols for extraction of viruses from various matrices and on the various specific detection techniques for each virus type.     

Adhesion of human pathogenic enteric viruses and surrogate viruses to inert and vegetal food surfaces

Enteric viruses, particularly human Noroviruses (NoV) and hepatitis A virus (HAV), are key food-borne pathogens. The attachment of these pathogens to foodstuff and food-contact surfaces is an important mechanism in the human contamination process. Studies were done to investigate the nature of the physicochemical forces, such as hydrophobic and electrostatic ones, involved in the interaction virus/matrix but, at this day, only few data are available concerning surface properties of viruses and prediction of the adhesion capacity of one specific virus onto matrices is still very difficult. The purpose of this study was to propose a reference system, including a representative virus surrogate, able to predict as close as possible behaviour of pathogenic viruses in term of adhesion on inert (stainless steel and polypropylene) and food surfaces (lettuce leaves, strawberries and raspberries). The adhesion of human pathogenic enteric viruses, cultivable strain of HAV and non-cultivable strains of human NoV (genogroups I and II), have been quantified and compared to these of human enteric viruses surrogates, included the MNV-1 and three F-specific RNA bacteriophages (MS2, GA and Qβ). A standardized approach was developed to assess and quantify viral adhesion on tested matrices after a contact time with each virus using real-time RT-PCR. Methods used for virus recovery were in accordance with the CEN recommendations, including a bovine Enterovirus type 1 as control to monitor the efficiency of the extraction process and amplification procedure from directly extracted or eluted samples. The adhesion of human pathogenic viruses, ranging from 0.1 to 2%, could be comparable for all matrices studied, except for NoV GII on soft fruits. Adhesion percentages obtained for the studied surrogate virus and phages were shown to be comparable to those of HAV and NoV on inert and lettuce surfaces. The MNV-1 appeared as the best candidate to simulate adhesion phenomena of all human pathogenic enteric viruses on all studied surfaces, while MS2 and GA bacteriophages could be a good alternative as model of viral adhesion on inert and lettuce surfaces. These results will be usable to design relevant experimental systems integrating adhesion behaviour of enteric viruses in the assessment of the efficiency of a technological or hygienic industrial process.     

Fate of Foodborne Viruses in the “Farm to Fork” Chain of Fresh Produce

Norovirus (NoV) and hepatitis A virus (HAV) are the most important foodborne viruses. Fresh produce has been identified as an important vehicle for their transmission. In order to supply a basis to identify possible prevention and control strategies, this review intends to demonstrate the fate of foodborne viruses in the farm to fork chain of fresh produce, which include the introduction routes (contamination sources), the viral survival abilities at different stages, and the reactions of foodborne viruses towards the treatments used in food processing of fresh produce. In general, the preharvest contamination comes mainly from soli fertilizer or irrigation water, while the harvest and postharvest contaminations come mainly from food handlers, which can be both symptomatic and asymptomatic. Foodborne viruses show high stabilities in all the stages of fresh produce production and processing. Low‐temperature storage and other currently used preservation techniques, as well as washing by water have shown limited added value for reducing the virus load on fresh produce. Chemical sanitizers, although with limitations, are strongly recommended to be applied in the wash water in order to minimize cross‐contamination. Alternatively, radiation strategies have shown promising inactivating effects on foodborne viruses. For high‐pressure processing and thermal treatment, efforts have to be made on setting up treatment parameters to induce sufficient viral inactivation within a food matrix and to protect the sensory and nutritional qualities of fresh produce to the largest extent.     

Norovirus, hepatitis A virus and enterovirus presence in shellfish from high quality harvesting areas in Portugal

This is the first report on the screening of shellfish from Portugal for the presence of human enteropathogenic viruses. Approximately 2000 shellfish (Curbicula fluminea, Ruditapes decussatus, Tellina crassa, Spisula solida, Dosinia exoleta, Ensis spp., Mytilus spp., Ostrea edulis and Cerastoderma edule), organized in 49 batches, were collected between March 2008 and February 2009. They were tested for norovirus (NoV), hepatitis A virus (HAV) and enterovirus (EV) by RT-PCR followed by nucleotide sequencing. Bacterial contamination was also evaluated by Escherichia coli counts. Viral contamination was detected throughout the year in all shellfish species and in all collection areas, independently of their harvesting areas classification. Overall, 67% of all analyzed batches were contaminated by at least one of the studied viruses while the simultaneous presence of two and three viruses was detected in 22% and 6% batches, respectively. Of the three viruses, NoV was detected in 37% of the batches, followed by EV in 35%, and HAV in 33%. Nucleotide sequencing of the NoV and HAV RT-PCR products demonstrated that all strains belonged to NoV genotype GII.4 and HAV subgenotype 1B. The presence of NoV and HAV in shellfish from “A class” harvesting areas of Portugal can represent a potential health risk.