北京市市售牡蛎中诺如病毒污染对居民健康影响的初步定量风险评估

目的通过建立针对北京市居民从零售到餐桌的牡蛎-诺如病毒暴露模型,对北京市居民通过市售牡蛎暴露诺如病毒的风险开展初步定量评估。方法利用北京市市售牡蛎中诺如病毒的356条定量检测数据,模拟北京市居民牡蛎消费情景,建立暴露评估模型,结合文献发表的基于暴发数据推导的剂量-反应关系模型,对居民通过生食牡蛎后发生诺如病毒感染的健康风险及其影响因素进行评估。结果 GI型和GII型诺如病毒阳性样品的污染水平分别为2.62×104个病毒拷贝/g(95%CI:3.73×103~1.54×105)和5.02×104个病毒拷贝/g(95%CI:8.13×103~2.52×105)。对血清分泌受体阳性的人群,生食1个可能受GI型和GII型诺如病毒污染的牡蛎的发病风险分别为0.93(95%CI:0.73~0.98)和0.95(95%CI:0.80~0.99);对血清分泌受体阴性的人群,生食1个可能污染GI型和GII型诺如病毒牡蛎的发病风险分别为0.37(95%CI:0.04~0.64)和0.57(95%CI:0.07~0.99)。风险的估计值与诺如病毒阴性样品的赋值相关,相关系数为0.49。不确定性分析显示,现有检测方法的检出限高于半数感染及致病剂量,是评估结果不确定性的主要来源。结论北京市居民通过生食牡蛎发生食源性诺如病毒感染的风险很高;提高病毒拷贝定量检测技术的灵敏度,降低检测方法的检出限水平是降低评估结果不确定性的主要途径。     

北京市市售牡蛎中诺如病毒核酸检测及定量分析

目的针对北京市市售牡蛎样品中诺如病毒污染水平及污染浓度进行定量分析研究。方法分离牡蛎消化腺,将消化腺匀浆处理,加入含有蛋白酶K的磷酸盐缓冲液,进行样品前处理,用试剂盒提取病毒RNA,用一步法实时荧光逆转录聚合酶链式反应(real time RT-PCR)检测诺如病毒RNA,并对阳性样品进行定量分析。结果共检测牡蛎样品356份,其中GGI阳性样品12份,GGII阳性样品39份,GGI和GGII同时为阳性的样品6份。对阳性样品中的诺如病毒核酸定量分析,核酸浓度在3.7×10~3~2.8×10~5基因拷贝/g(消化腺)之间。结论北京市市售牡蛎中存在诺如病毒污染的情况,需要加强对牡蛎中诺如病毒的污染监测,并开展污染水平风险评估,保障消费者食用安全,降低由诺如病毒引起的腹泻病的疾病负担。     

广西养殖牡蛎中诺如病毒的污染状况及风险评估

目的研究广西养殖场、农贸市场及餐饮场所牡蛎中诺如病毒的污染状况,对广西养殖牡蛎中诺如病毒可能引发的发病风险进行评估。方法采用荧光逆转录-聚合酶链式反应(RT-PCR)法对广西养殖场、农贸市场、餐饮场所牡蛎样品中诺如病毒污染状况进行检测,采用Risk Ranger软件对牡蛎中诺如病毒进行半定量风险评估。结果 480份牡蛎样品中诺如病毒总检出率为11.04%(53/480),其中养殖场及农贸市场检出率分别为15.83%(38/240)、12.50%(15/120),餐饮场所牡蛎样品未检出诺如病毒;基因分型结果显示检出的诺如病毒均为GⅡ型。风险评估结果显示,加工后食用和生食的风险评分为44和67分,分别为中度和高度风险,预计食用者每人每天患病的可能性分别为3.29×10~(-6)和3.29×10~(-2),预计广西每年患病人数分别为3.10×10~3和3.10×10~7人。结论广西养殖场及农贸市场牡蛎中诺如病毒污染情况较为严重,污染的诺如病毒基因型均为GⅡ型,不生食牡蛎及食用前有效的加工处理是减少诺如病毒食源性疾病发生的有效方法。     

西生菜中低污染量GⅡ型诺如病毒的富集与定量检测研究

目的优化新鲜西生菜中低污染量的GⅡ型诺如病毒富集方法,比较实时荧光RT-PCR与微滴式数字RT-PCR检测方法,研究适用于新鲜西生菜中低污染量的GⅡ型诺如病毒的定量检测方法。方法选取洗脱液种类、沉淀剂种类、沉淀剂作用温度及沉淀剂作用时间4个因素进行研究,以确定较优的GⅡ型诺如病毒富集条件。结合较优的富集条件,分别采用实时荧光RT-PCR方法和微滴式数字RT-PCR方法进行定量检测。结果病毒富集方法研究表明:洗脱液为牛肉膏-甘氨酸洗脱液、沉淀剂为PEG6000+PEG8000、沉淀剂作用温度为4℃,沉淀剂作用时间为4 h为较优条件,此时富集后的平均病毒浓度最高,平均回收率可达53.43%。实时荧光RT-PCR的检测限为1.44×10~4 copies/g;而微滴式数字RT-PCR的检测限为7.86×10~2 copies/g。结论通过优化西生菜中低污染量的GⅡ诺如病毒富集方法,比较实时荧光RT-PCR方法与微滴式数字RT-PCR方法定量检测病毒的效果,建立了适用于西生菜样品中低污染量GⅡ诺如病毒的定量检测方法。     

2018—2022年淄博市市售双壳贝类中诺如病毒检测分析

目的：分析淄博市市售双壳贝类中诺如病毒检测结果,了解其污染情况,为开展食品中诺如病毒风险评估及采取有效防控措施提供数据支持。方法：采集2018—2022年淄博市市售双壳贝类样本155份,提取病毒核酸并采用实时荧光定量逆转录聚合酶链式反应分别进行诺如病毒GI型和GII型检测。结果：2018—2022年采集市售双壳贝类155份,检出诺如病毒阳性29份,总阳性检出率为18.71%。GII型阳性检出率（10.97%）高于GI型阳性检出率（4.52%）,差异有统计学意义（P <0.05）,不同季度检出率差异有统计学意义（P <0.05）,第一季度检出率最高（50.00%）。结论：淄博市市售双壳贝类中存在诺如病毒污染现象,以GII型为主,第一季度检出率最高,需加强监测,开展相关风险评估,以降低诺如病毒引起的食源性疾病负担。     

甘肃省兰州地区贝类海鲜、蔬菜水果中诺如病毒污染监测分析

目的:掌握甘肃省兰州地区海鲜贝类、蔬菜水果等诺如病毒的污染状况,发现食品安全隐患。方法:于2017年6月—2018年3月间,在兰州地区批发市场、零售市场、超市随机抽取120个贝类、蔬菜、水果样品,基于Taq Man探针法原理,通过特异性引物、探针结合,采用实时荧光RT-PCR技术对样品进行了GI和GII基因型诺如病毒的检测。结果:诺如病毒总阳性率为4.17%,其中GI型诺如病毒基因不存在,均为GII型。结论:该研究对了解兰州地区贝类海鲜、蔬菜水果中诺如病毒污染状况,加强诺如病毒预防控制提供了科学依据。     

2011—2012年广东省市售牡蛎中诺如病毒污染调查分析

目的 调查2011-2012年广东省市售牡蛎中诺如病毒的污染状况,为采取有效措施减轻牡蛎中诺如病毒污染程度提供建议,达到预防和控制食用牡蛎引起诺如病毒胃肠炎疾病的目的.方法 2011年3月-2012年10月,在广东省部分沿海城市进行市售牡蛎的采样检测,对其诺如病毒污染状况进行连续两年监测,采用荧光RT-PCR检测诺如病毒阳性标本基因分型,并对不同城市、季节及场所牡蛎中诺如病毒的污染情况及基因分型情况进行比较.结果 牡蛎中诺如病毒总检出率为14.9％ (41/275);四个季节牡蛎中诺如病毒检出率依次为4.4％、15.7％、18.2％和36.7％;从基因分型分析,GⅠ型病毒株检出率为4.0％,GⅡ型病毒株检出率为6.2％,GⅠ和GⅡ型病毒株同时检出率为4.7％.结论 2011-2012年广东省市售牡蛎的诺如病毒污染情况在市场、餐饮场所以及不同地区间比较差异无统计学意义,其基因分型间比较差异无统计学意义,但呈现明显季节性特点,以冬季污染最严重.     

草莓中诺如病毒和甲肝病毒的多重实时荧光逆转录PCR检测方法的建立

目的建立草莓中诺如病毒GI、诺如病毒GII和甲肝病毒等3种食源性病毒的多重实时荧光RT-PCR检测方法,并应用于实际样品检测。方法对草莓样品进行前处理、病毒富集、病毒RNA提取和纯化后,先采用单重实时荧光RT-PCR进行检测,随后进行多重实时荧光RT-PCR反应条件优化,建立多重实时荧光RT-PCR检测方法并分析其特异性和灵敏度。结果所采用的病毒富集和核酸提取方法可以实现病毒的有效富集和抑制剂的去除,建立的多重实时荧光RT-PCR方法特异性强(100%),对草莓样品中诺如病毒GI、诺如病毒GII和甲肝病毒的检测灵敏度分别为56.2 RT-PCR50/20 g、31.6 RT-PCR50/20 g和31.4 CCID50/20 g。同时对50份样品进行检测,结果均为阴性。结论所建立的检测方法快速、灵敏、特异性强,适用于草莓产品中诺如病毒GI、诺如病毒GII和甲肝病毒的同时检测。     

福建省养殖环节牡蛎中诺如病毒污染状况分析

目的 了解福建省养殖环节牡蛎中诺如病毒(Norovirus)污染状况及基因分型,进一步定量分析诺如病毒污染水平,为食品中诺如病毒污染监测及安全风险评估提供数据支持.方法 2017年8月—2018年9月,于福建省某养殖基地采集牡蛎样品共244份,采用实时荧光定量聚合酶链式反应(PCR)法对样品中诺如病毒进行检测,确定其污...     

Surveillance of Enteric Viruses and Microbial Indicators in the Eastern Oysters (Crassostrea virginica) and Harvest Waters along Louisiana Gulf Coast

Noroviruses are the most common causative agent of viral gastroenteritis in humans, and are responsible for major foodborne illnesses in the United States. Filter‐feeding molluscan shellfish exposed to sewage‐contaminated waters bioaccumulate viruses, and if consumed raw, transmit the viruses to humans and cause illness. We investigated the occurrence of norovirus GI and GII and microbial indicators of fecal contamination in the eastern oysters (Crassostrea virginica) and water from commercial harvesting areas along the Louisiana Gulf Coast (January to November of 2013). Microbial indicators (aerobic plate count, enterococci, fecal coliforms, Escherichia coli, male‐specific coliphages, and somatic coliphages) were detected at the densities lower than public health concerns. Only one oyster sample was positive for norovirus GII at 3.5 ± 0.2 log₁₀ genomic equivalent copies/g digestive tissues. A stool specimen obtained from an infected individual associated with a norovirus outbreak and the suspected oysters (Cameron Parish, La., area 30, January 2013) were also analyzed. The norovirus strain in the stool belonged to GII.4 Sydney; however, the oysters were negative and could not be linked. In general, no temporal trend was observed in the microbial indicators. Low correlation among bacterial indicators was observed in oysters. Strongest correlations among microbial indicators were observed between enterococci and fecal coliforms (r = 0.63) and between enterococci and E. coli (r = 0.64) in water (P < 0.05); however, weak correlations were found in oysters (r < 0.45) and between oysters and harvest water (r ≤ 0.36, P > 0.05). Our results emphasize the need for regular monitoring of pathogenic viruses in commercial oyster harvesting areas to reduce the risks of viral gastroenteritis incidences.     

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。     

Comparison of norovirus RNA levels in outbreak-related oysters with background environmental levels

Norovirus is the principal agent of bivalve shellfish-associated gastroenteric illness worldwide. Numerous studies using PCR have demonstrated norovirus contamination in a significant proportion of both oyster and other bivalve shellfish production areas and ready-to-eat products. By comparison, the number of epidemiologically confirmed shellfish-associated outbreaks is relatively low. This suggests that factors other than the simple presence or absence of virus RNA are important contributors to the amount of illness reported. This study compares norovirus RNA levels in oyster samples strongly linked to norovirus or norovirus-type illness with the levels typically found in commercial production areas (non-outbreak-related samples). A statistically significant difference between norovirus levels in the two sets of samples was observed. The geometric mean of the levels in outbreak samples (1,048 copies per g) was almost one order of magnitude higher than for positive non-outbreak-related samples (121 copies per g). Further, while none of the outbreak-related samples contained fewer than 152 copies per g, the majority of positive results for non-outbreak-related samples was below this level. These observations support the concept of a dose-response for norovirus RNA levels in shellfish and could help inform the establishment of threshold criteria for risk management.     

Rapid virus detection procedure for molecular tracing of shellfish associated with disease outbreaks

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III-spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.     

Determination of norovirus contamination in oysters from two commercial harvesting areas over an extended period, using semiquantitative real-time reverse transcription PCR

The human health risk associated with the consumption of molluscan shellfish grown in sewage-contaminated waters is well established. Noroviruses, which cause gastroenteritis, are the principal agents of shellfish-related illness. Fecal-indicator quality standards based on Escherichia coli are well established in Europe and elsewhere. However, norovirus outbreaks after consumption of shellfish meeting these standards still occur, and the need to improve consumer health protection is well recognized. Alternative approaches proposed include direct monitoring of viral pathogens and the use of alternative indicator organisms capable of providing a better indication of virus risk. This study applies a recently developed TaqMan PCR assay to assess norovirus contamination in shellfish. Comparison was made with E. coli as the existing sanitary standard and a male-specific RNA bacteriophage as a possible alternative. Two commercial pacific oyster (Crassostrea gigas) harvesting areas were monitored over a 31-month period. The results show peaks of norovirus contamination in both areas during winter months, with average levels approximately 17 times higher in oysters sampled October to March than during the remainder of the year, consistent with epidemiological data for the United Kingdom showing oyster-associated illness is confined to winter months. While there was no apparent association with E. coli, an association between levels of norovirus contamination and the male-specific RNA bacteriophage was noted, with average norovirus levels over 40 times higher in samples with male-specific RNA bacteriophage counts of >1,000 PFU/100 g than in samples with <100 PFU/100 g. Overall, these results suggest that norovirus monitoring in shellfish production areas could be an effective strategy for reduction of virus risk.