Evaluation of a norovirus detection methodology for ready-to-eat foods

Despite recent norovirus (NoV) foodborne outbreaks related to consumption of ready-to-eat (RTE) foods, a standardized assay to detect NoV in these foods is not available yet. Therefore, the robustness of a methodology for NoV detection in RTE foods was evaluated. The NoV detection methodology consisted of direct RNA extraction with an eventual concentration step, followed by RNA purification and a multiplex RT-qPCR assay for the detection of GI and GII NoV and the murine norovirus-1 (MNV-1), the latter used as process control. The direct RNA extraction method made use of the guanidine-isothiocyanate containing reagent (Tri-reagent?, Ambion) to extract viral RNA from the food sample (basic protocol called TriShort), followed by an eventual concentration step using organic solvents (extended protocol called TriConc). To evaluate the robustness of the NoV detection method, the influence of (1) the NoV inoculum level and (2) different food types on the recovery of NoV from RTE foods was investigated. Simultaneously, the effect of two RNA purification methods (manual RNeasy minikit (Qiagen) and automated NucliSens EasyMAG (BioMérieux)) on the recovery of NoV from these foods was examined. Finally, MNV-1 was evaluated as process control. First of all, high level GI and GII NoV inocula (~10? NoV genomic copies/10 g) could be recovered from penne salad samples (10 g) in at least 4 out of 6 PCRs, while low level GI and GII NoV inocula (~10? NoV genomic copies/10 g) could be recovered from this food product in maximally 3 out 6 PCRs, showing a significant influence of the NoV inoculum level on its recovery. Secondly, low level GI and GII NoV inocula (10? NoV genomic copies/10 g) were spiked onto 22 ready-to-eat food samples (10 g) classified in three categories (soups, deli sandwiches and composite meals). The GI and GII NoV inocula could be recovered from 20 of the 22 samples. The TriConc protocol provided better recoveries of GI and GII NoV for soups while the TriShort protocol yielded better results for the recovery of GII NoV from composite meals. NoV recovery from deli sandwiches was problematic using either protocol. Thirdly, the simultaneous comparison of two RNA purification protocols demonstrated that automated RNA purification performed equally or better compared to manual RNA extraction. Finally, MNV-1 was successfully evaluated as process control when detecting NoV in RTE foods using this detection methodology. In conclusion, the evaluated NoV detection method was capable of detecting NoV in RTE foods, although recoveries were influenced by the inoculum level and by the food type.     

Genetic variation in the conservative gene region of Norovirus genogroup II strains in environmental and stool samples

Noroviruses (NoVs) have been one of leading etiological agents for infectious gastroenteritis over the world. Gastroenteritis caused by NoVs is prevalent in winter season, and the contamination of the water environment with NoVs in the epidemic cold season is frequently reported. In contrast, the number of gastroenteritis patients and NoVs in the water environment are reduced during the nonepidemic summer season, and the year-round fate of NoVs has remained to be elucidated. In this study, we collected nucleotide sequences of NoV genogroup II (GII) from domestic sewage, sewage sludge, treated wastewater, river water, and stool samples of gastroenteritis patients in geographically close areas. Phylogenetic analysis of the obtained NoV gene revealed that six out of seven isolates from environmental samples and 10 out of 11 isolates from stool samples belong to genotype 3 (NoV GII.3) or 4 (NoV GII.4), which have been prevalent throughout the world. Genetic distances between the conservative gene region of NoV GII.4 variants implied that genetically diverse strains are likelyto occur in environmental samples. The evaluation of the evolutionary change of NoV gene obtained from environmental samples would make it possible to elucidate the year-round fate of NoVs.     

Detection of GI and GII noroviruses in drinking water and vegetables using filtration and real-time RT-PCR

The purpose of the study was to provide a rapid and sensitive method for detecting NoV GI and NoV GII in drinking water and vegetables. The method is based on viral concentration by microporous membrane adsorption method before RNA extraction and real-time RT-PCR amplification. Then water and vegetable samples which artificially contaminated with NoV GI and GII stool samples were used to determine the mean virus recoveries and the method sensitivity. The method showed the detection limit of NoV GI was 4.13 × 10² copies/500 mL for drinking water and 4.13 × 10³ copies/15 g for lettuce and coriander. The detection limit of NoV GII was 2.94 × 10¹ copies/500 mL for distilled water, 2.94 × 10² copies/500 mL for Mountain spring water and mineral water, and 2.94 × 10³ copies/15 g for lettuce and coriander. The method described provides a valuable tool for monitoring the potential public health risks associated with noroviruses contamination in drinking water and vegetables.     

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。     

实时荧光RT-PCR检测贝类中的札幌病毒

建立检测贝类中GⅠ、GⅡ、GⅣ和GⅤ型札幌病毒的实时荧光RT-PCR新方法。首先使用PEG 8000对贝类中的札幌病毒进行富集,然后采用Tri-reagent提取材料中的总RNA,针对札幌病毒RNA 3'端含Poly A尾的特点,使用带有Poly(dT)25的磁珠对病毒RNA进行纯化,用所获的高纯度RNA进行四种型别札幌病毒的实时荧光RT-PCR检测。该方法高效、灵敏,检测下限为101数量级拷贝,能够用于日常检验。     

Statistical analysis of attack rate in norovirus foodborne outbreaks

Norovirus (NoV), which causes foodborne gastroenteritis outbreaks, is one of the important viruses in public health. We statistically analyzed the attack rate in foodborne outbreaks caused by NoV. The attack rate in 95 oyster-associated outbreaks was significantly higher than that in 195 food handler-associated outbreaks (P=0.007). The difference in the number of NoV genotypes implicated is considered to be an important factor for this difference. The attack rate in 20 outbreaks associated only with GII/3 was higher than that in 143 other outbreaks (P=0.247), while the attack rate in 27 outbreaks associated only with GII/4 was lower than that in 136 other outbreaks (P=0.004), suggesting that GII/4 NoVs cause asymptomatic infection more frequently than do other NoV genotypes. Our results suggest that differences in implicated foods, susceptibility of the host to NoV infection, and pathogenicity of NoVs may influence the attack rate in NoV foodborne outbreaks.     

RT-PCR法检测贝类中的甲肝病毒的研究

在世界范围内,甲肝病毒是与食用贝类有关的主要传染性疾病之一。由于贝类中含有PCR抑制剂以及病毒富集过程中病毒的回收率低,阻碍了天然污染的贝类中HAV的PCR检测。研究中建立了一种经苷氨酸缓冲液洗涤,2次PEG沉降富集病毒,然后进行RNA提取和RT-PCR对贝类中的甲肝病毒进行检测的方法。经比较,采用小体系肠道样品检测比采用全贝检测的富集效果更佳,并比较了PEG8000和PEG6000对病毒富集的效果,回收率分别为13.5%和7.6%,此方法可有效地降低PCR抑制剂的影响,最低检测限可达10个TCID50/1.5 g。     

Enhanced immunomagnetic separation for the detection of norovirus using the polyclonal antibody produced with human norovirus GII.4-like particles

Human norovirus (NoV) is the most common cause of foodborne viral gastroenteritis worldwide. This study was aimed to develop the enhanced immunomagnetic separation (IMS) for effectively concentrating and detecting human noroviruses in food matrix. Virus-like particles (VLPs) were made by integrating NoV GII.4 capsid gene into baculovirus vector. In order to increase the sensitivity and specificity of immunomagnetic complex, polyclonal rabbit antibody against NoV GII.4 capsid was produced and used for producing immunomagnetic beads. IMS, polyethylene glycol precipitation, and ultrafiltration were compared to concentrate NoV spiked in vegetables. IMS was the most efficient method for concentrating NoV. Therefore, IMS developed in this study is the most effective method to concentrate and detect NoV contaminated in produce.     

实时荧光RT-PCR检测冷冻草莓中诺如病毒

目的：建立冷冻草莓中的GI、GII型诺如病毒实时荧光RT-PCR检测方法,并应用于实际样品的检测。方法：对草莓样品进行前处理、病毒富集、病毒RNA的提取和纯化,然后采用实时荧光RT-PCR进行检测。结果：核酸提取方法能够有效地去除抑制因子,同时对104份送检样品进行检测,结果均为阴性。结论：所建立的核酸提取与实时荧光RT-PCR结合的检测体系适合于草莓样品中诺如病毒GI、GII型的检测。