贻贝中诺如病毒荧光定量反转录PCR检测及北京市污染情况调查

目的监测北京市市售贻贝中诺如病毒(NoV)的污染率及污染浓度,为贝类样品中诺如病毒的风险评估提供基础数据。方法解剖并分离贻贝的内脏团,将内脏团研磨至均质,加入磷酸盐缓冲液振荡60 min,提取病毒颗粒。提取病毒RNA,荧光定量反转录PCR检测诺如病毒,并进行定量分析。结果共检测293份贻贝样品,总阳性率为9.22%(27/293),其中诺如病毒基因组Ⅰ(GGⅠ)占阳性样品数的37.04%(10/27),基因组Ⅱ(GGⅡ)占阳性样品数的62.96%(17/27),未检测到同时携带GGⅠ和GGⅡ的样品。阳性样品中诺如病毒浓度范围在6.20×10~3~3.15×10~5基因拷贝/g(内脏)之间。结论北京市市售贻贝中存在诺如病毒污染。     

The efficacy of EO waters on inactivating norovirus and hepatitis A virus in the presence of organic matter

The ability of acidic electrolyzed oxidizing water (AEO) and neutral electrolyzed oxidizing water (NEO) to inactivate the murine norovirus (MNV-1) surrogate for human norovirus and hepatitis A virus (HAV) in suspension and on stainless steel coupons in the presence of organic matter was investigated. Viruses containing tryptone (0.0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%) were mixed with AEO and NEO for 1 min. In addition, stainless steel coupons containing MNV-1 with or without organic matter were treated with AEO or NEO for 3, 5, and 10 min. AEO was proven effective and generally killed more MNV-1 and HAV in suspension than NEO. Depending on the EO water generator, free chlorine concentrations are required to inactivate MNV-1 and HAV by 3-log PFU/mL or greater ranged from 30 mg/L to 40 mg/L after a 1 min contact time. The virucidal effect increased with increasing free chlorine concentration and decreased with increasing tryptone concentration in suspension. Both AEO and NEO at 70–100 mg/L of free chlorine concentration significantly reduced MNV-1 on coupons in the absence of organic matter. However, there was no significant difference between these two treatments in the presence of organic matter. In addition, the efficacy of these two EO waters on stainless steel coupons increased with the increasing treatment time. Results indicated that AEO and NEO can reduce MNV-1 and HAV in suspension. However, higher free chlorine concentrations and longer treatment times may be necessary to reduce viruses on contact surfaces or in the presence of organic matter.     

Transmission of Common Foodborne Viruses by Meat Products

The most common foodborne viruses are single stranded RNA viruses which are adaptable and extremely resistant to environmental stress factors. Usual routes of food contamination are via stool material by persons shedding intestinal virus, or by saliva aerosols generated by shedding persons when coughing. Contamination of meat by animal viruses occurs when good hygienic and manufacturing practice fails. Once within food, viruses cannot replicate since they require living cells for this; hence food is not sensorily altered. Preventive measures in meat processing against pathogenic bacteria frequently have poor antiviral performance, while diagnostic techniques for viruses remain problematic.     

Foodborne and Waterborne Pathogenic Bacteria in Selected Organisation for Economic Cooperation and Development (OECD) Countries

The World Ranking Food Safety Performance reports by Charlebois in 2008 and 2010 importantly stimulated international discussion and encouraged efforts to establish realistic international benchmarks for food safety performance among Organisation for Economic Cooperation and Development (OECD) countries. This paper presents the international incidence of 5 common foodborne pathogens and describes the challenges of comparing international data. Data were compiled from surveillance authorities in the countries, such as the Natl. Notifiable Diseases Surveillance System of Australia; the Canadian Notifiable Diseases Surveillance System; the European Food Safety Authority, EFSA; the Ministry of Health, Labour and Welfare of Japan; New Zealand Food Safety Authority; and the U.S. Center for Disease Control and Prevention. The highest average rates in cases per 100000 people over the 12‐y period from 2000 to 2011 for Campylobacter spp. (237.47), Salmonella spp. (67.08), Yersinia spp. (12.09), Verotoxigenic/Shiga toxin producing Escherichia coli (3.38), and Listeria monocytogenes (1.06) corresponded, in order, to New Zealand, Belgium, Finland, Canada, and Denmark. Comparatively, annual average rates for these 5 pathogens showed an increase over the 12‐y period in 28%, 17%, 14%, 50%, and 6% of the countries for which data were available. Salmonella spp. showed a decrease in 56% of the countries, while incidence of L. monocytogenes was constant in most countries (94%). Variable protocols for monitoring incidence of pathogens among OECD countries remain. Nevertheless, there is evidence of sufficient standardization of monitoring protocols such as the European Surveillance System, which has contributed to reduce this gap.     

Seasonal and regional prevalence of norovirus,hepatitis A virus,hepatitis E virus,and rotavirus in shellfish harvested from South Korea

Foodborne viruses such as norovirus (NoV), hepatitis A virus (HAV), hepatitis E virus (HEV), and rotavirus (RoV) are transmitted through water and food contaminated with stool. The purpose of this study was to examine the prevalence of foodborne viruses in shellfish collected in South Korea using real-time RT-PCR. Virus was eluted from the stomach and the digestive diverticula of 152 shellfish (51 oysters, 51 Manila clams, and 50 mussels) and concentrated with polyethylene glycol precipitation. The detection rate of NoV genogroup II, NoV genogroup I, HAV, HEV, and RoV was 21.7%, 5.9%, 0.7%, 0%, and 0% of shellfish, respectively. Although the geographic distribution of NoV was statistically significant, prominent seasonal variation in NoV was not observed in this study. In order to reduce norovirus food poisoning in the public, it is important to prevent the contamination of NoV in shellfish in South Korea.     

Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters

Norovirus (NoV), the leading cause of adult non-bacterial gastroenteritis can be commonly detected in wastewater but the extent of NoV removal provided by wastewater treatment plants (WWTPs) is unclear. We monitored a newly commissioned WWTP with UV disinfection on a weekly basis over a six month period for NoV using RT-qPCR and for FRNA bacteriophage GA using both RT-qPCR (total concentration) and a plaque assay (infectious concentration). Mean concentrations of NoV GI and GII in influent wastewater were reduced by 0.25 and 0.41 log₁₀ genome copies 100 ml⁻¹, respectively by the WWTP. The mean concentration of total FRNA bacteriophage GA was reduced by 0.35 log genome copies 100 ml⁻¹ compared to a reduction of infectious FRNA bacteriophage GA of 2.13 log PFU 100 ml⁻¹. A significant difference between concentrations of infectious and total FRNA bacteriophage GA was observed in treated, but not in untreated wastewaters. We conclude that RT-qPCR in isolation underestimates the reduction of infectious virus during wastewater treatment. We further compared the concentrations of infectious virus in combined sewer overflow (CSO) and UV treated effluents using FRNA bacteriophage GA. A greater percentage (98%) of infectious virus is released in CSO discharges than UV treated effluent (44%). Following a CSO discharge, concentrations of NoV GII and infectious FRNA bacteriophage GA in oysters from less than the limit of detection to 3150 genome copies 100 g⁻¹ and 1050 PFU 100 g⁻¹ respectively.     

2017年北京市哨点医院腹泻病例诺如病毒感染状况流行病学特征分析

目的 了解2017年北京市食源性疾病监测哨点医院腹泻病例诺如病毒感染的流行病学特征,为诺如病毒的预防控制提供科学依据。方法 采用描述性流行病学方法对收集的北京市35家哨点医院腹泻病例(2 490例)的流行病学信息和诺如病毒检测结果进行统计分析。结果 2 490份病例标本中,诺如病毒的检出率为14.30%(356/2 490),以GⅡ基因组为主(87.08%,310/356)。第一季度和第二季度的诺如病毒检出率较高,分别为17.77%(99/557)和20.69%(149/720);不同性别腹泻病例诺如病毒检出率差异无统计学意义(P＞0.05);不同年龄组腹泻病例诺如病毒检出率差异有统计学意义(P＜0.05),且以15～24岁年龄组检出率最高(21.66%,81/374)。诺如感染者出现恶心(18.40%,188/1 022)和呕吐(22.87%,145/634)症状的比例要明显高于其他腹泻病例。结论 2017年北京市食源性疾病监测哨点医院腹泻病例诺如病毒感染以GⅡ基因组为主,散发感染的流行高峰出现在第一和第二季度,青年人群检出率较高。     

餐饮服务单位及宾馆住宿场所环境和食品从业人员中诺如病毒感染状况研究

目的了解诺如病毒在餐饮服务单位和宾馆住宿场所环境中的分布及厨师隐性感染状况,并对其基因型别进行研究。方法共抽检北京市西城区40家餐饮服务单位和10家宾馆住宿场所,每家单位采集4份环境涂抹样品及2名厨师粪便标本。用实时荧光聚合酶链式反应(PCR)对诺如病毒进行初步鉴定。通过逆转录聚合酶链式反应(RT-PCR)对诺如病毒开放读码框(opening reading frames,ORF)1区序列进行扩增,并对测序结果进行序列和进化分析。结果共采集环境表面涂抹样品200份,厨师粪便标本100份。其中,1份墩布池涂抹样品为诺如病毒阳性,环境样品诺如病毒阳性率为0.5%(1/200),3份厨师粪便中检出诺如病毒,隐性感染率为3.0%(3/100)。3名厨师粪便标本诺如病毒ORF1区序列经RT-PCR扩增,进化树显示,1名厨师为GII.17基因型,2名厨师为GI.3基因型。结论北京市西城区餐饮服务单位及宾馆住宿场所存在诺如病毒传播风险,应强化卫生管理,重视物体表面消毒及厨师健康教育,防止诺如病毒的进一步扩散。     

GⅠ/GⅡ型诺如病毒两联装甲RNA标准样品的研制

针对目前缺乏适配多项检测标准、稳定、安全的诺如病毒RNA标准样品的问题,研制基于MS2噬菌体内含常见GⅠ/GⅡ型诺如病毒检测靶标两联装甲RNA标准参考样品。人工合成MS2噬菌体成熟酶基因、衣壳蛋白基因、包装位点及GⅠ/GⅡ型诺如病毒靶标基因,克隆于表达载体pET-28a(+)中,构建重组质粒p ET-MS2-NoV。经大肠杆菌BL21诱导表达,先后利用PEG6000、酶处理和丙烯葡聚糖凝胶层析柱纯化表达产物。SDS-PAGE和透射电镜鉴定产物大小及结构,荧光定量PCR检测有无残留核酸。之后对纯化的病毒样颗粒(Virus-like particles,VLPs)开展定值、均匀性和短期稳定性研究。SDS-PAGE结果表明重组质粒在BL21中表达出了目的蛋白,大小在10~15ku之间,与预期一致;纯化后的VLPs无杂蛋白和残留核酸;透射电镜下呈结构完整、大小均一的球状,直径约25 nm。纯化后AR-NoV中GⅠ型和GⅡ型靶标定值结果分别为(4.04±0.62)×10⁷ copies/μL和(6.16±0.30)×10⁷ copies/μL。单因素方差检验证实样品均一性良好,F相似文献