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.     

Evaluation of an Extraction Method for the Detection of GI and GII Noroviruses in Fruit and Vegetable Salads

Human norovirus (HuNoV) is a major foodborne virus causing gastroenteritis outbreaks in humans. Salad products can be vectors of transmission for foodborne viruses such as HuNoV when these products are contaminated naturally or through unsanitary food handling. Therefore, development of simple, reliable and sensitive techniques for the detection of HuNoV in salad products is needed to ensure food safety. The purpose of our study was to optimize a method for the detection of HuNoV in artificially contaminated salad products. To this end, 2 different kinds of salads (fruit salads and vegetable salads) were experimentally inoculated with HuNoV GI, HuNoV GII, and MS2 suspensions. The selected method was based on treatment with pectinase followed by Trizol‐chloroform purification, and the recovery efficiencies were 6.07% to 26.52% for HuNoV GI and 5.54% to 37.36% for HuNoV GII. MS2 was used as the process control, and the recovery efficiencies for fruit salad and vegetable salad samples were 38.57% and 41.13%, respectively. The optimized method could be applied in diagnostic laboratories to identify NoV contamination in composite foods, such as salad products, should an event of foodborne outbreak occur.     

微滴式数字PCR检测冷冻草莓中GⅠ、GⅡ型诺如病毒

目的:建立一种检测冷冻草莓中诺如病毒(GⅠ和GⅡ)的逆转录微滴数字PCR (RT-ddPCR)方法。方法:根据ISO标准选定检测引物,优化反应体系,退火温度,进行了方法学实验,建立了一种快速检测冷冻草莓中GⅠ和GⅡ亚型诺如病毒的新方法。结果:确定了数字PCR检测GⅠ型诺如病毒退火温度为56.5℃,GⅡ型诺如病毒退火温度为58.1℃。RT-ddPCR检测GⅠ质粒标准品标准曲线的R2=0.9947,RT-ddPCR检测GⅡ质粒标准品标准曲线的R2=0.9950,说明该方法具有良好的线性关系。与RT-qPCR灵敏度对比,RT-ddPCR法的灵敏度比RT-qPCR法高一个数量级。在检测范围内,最低检测限低至个位拷贝数。RSD最小为3.8%,表明该实验重复性良好。浓度较低100 copies/μL左右时,RT-ddPCR的重复性不佳。结论:本研究建立的诺如病毒数字PCR法具有特异性强、灵敏度高、检测限低等优点,可用于冷冻草莓中诺如病毒的定量检测。     

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。     

The reduction of murine norovirus 1, B. fragilis HSP40 infecting phage B40-8 and E. coli after a mild thermal pasteurization process of raspberry puree

Pasteurization processes of raspberry puree are nowadays limited to short times and rather low temperatures to maintain flavor and nutritional quality. Norovirus (NoV) outbreaks associated with raspberries highlight the need to determine the survival of NoV on this type of soft fruit. Therefore, resistance of murine norovirus 1 (MNV-1), a surrogate for human NoV, B. fragilis HSP40 infecting phage B40-8, and E. coli towards mild pasteurization was tested. Raspberry puree heat treated at 65 degrees C for 30s showed a 1.86, 2.77, and 3.89 log reduction of, respectively, MNV-1, E. coli, and B40-8. Heating at 75 degrees C for 15s established a 2.81 log reduction of MNV-1 while a 3.44 and 3.61 log reduction of B40-8 and E. coli was observed. No supplementary lethal effect of holding the heat-treated raspberry puree at 4 degrees C overnight was noticed. B40-8 failed to be useful as a tool to monitor NoV inactivation during mild pasteurization processes. Moreover, <3 log reductions of MNV-1 were observed suggesting that upon high initial contamination load, infectious NoV particles may remain on mildly pasteurized raspberry puree.     

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。     

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.     

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.