RNA病毒检测质控物质-大肠杆菌噬菌体MS2标准样品的研制和应用

目的研制大肠杆菌噬菌体MS2标准样品,以其作为RNA病毒检测质控物质,建立RNA病毒检测全过程的质量控制体系。方法利用液体培养法制备大肠杆菌噬菌体MS2标准样品;采用双层琼脂法对标准样品进行稳定性、均匀性测定并定值;将标准样品添加于贝类样品中,应用于贝类诺如病毒实时荧光检测全过程质量控制。结果该标准样品稳定性与均匀性良好,定值为(1.57±0.0288)×10~(11) pfu/m L;保质期为12个月;在4种不同贝类样品中,病毒提取效率在3.70%~7.84%之间,符合国际标准ISO 15216:2-2013的病毒提取效率大于1%的要求。结论该研究制备的大肠杆菌噬菌体MS2标准样品可以对RNA病毒检测全过程进行良好的质量控制,具有良好的应用前景。     

Grape seed extract for foodborne virus reduction on produce

Grape seed extract (GSE) is reported to have antibacterial properties with few current studies on antiviral activity. Recently, we reported the effects of GSE against foodborne viral surrogates in vitro. This study evaluated the application of GSE (commercial Gravinol-S) against hepatitis A virus (HAV) and human norovirus surrogates, feline calicivirus (FCV-F9) and murine norovirus (MNV-1), on model produce. Washed and air-dried lettuce (3 × 3 cm²) and jalapeno peppers (25–30 g) were inoculated with FCV-F9, MNV-1, or HAV at high (∼7 log₁₀ PFU/ml) or low (∼5 log₁₀ PFU/ml) titers, and treated with 0.25, 0.5, 1 mg/ml GSE or water for 30 s to 5 min. Treatments were stopped/diluted with cell-culture media containing 10% heat-inactivated fetal bovine serum and evaluated using plaque assays. At high titers, FCV-F9 was reduced by 2.33, 2.58, and 2.71 log₁₀ PFU on lettuce; and 2.20, 2.74, and 3.05 log₁₀ PFU on peppers after 1 min using 0.25, 0.50, and 1 mg/ml GSE, respectively. Low FCV-F9 titers could not be detected after 1 min at all three GSE concentrations. Low titer MNV-1 was reduced by 0.2–0.3 log₁₀ PFU on lettuce and 0.8 log₁₀ PFU on peppers, without reduction of high titer. GSE at 0.25–1 mg/ml after 1 min caused 0.7–1.1 and 1–1.3 log₁₀ PFU reduction for high and low HAV titers, respectively on both commodities. Instrumental color analysis showed no significant differences between treated and untreated produce. GSE shows potential for foodborne viral reduction on produce as part of hurdle technologies.     

Comparison of chlorine and peroxyacetic-based disinfectant to inactivate Feline calicivirus, Murine norovirus and Hepatitis A virus on lettuce

In recent years, raw fruits and vegetables have frequently been involved in foodborne transmission to humans of enteric viruses, particularly noroviruses and hepatitis A virus (HAV). Although viral contamination can occur during all steps of food processing, primary production is a critical stage on which prevention measures must be focused to minimize the risk of infection to consumers. Postharvest sanitation may be a valid technological solution for decreasing the bacterial load on fresh raw material, but there is a lack of data concerning the effectiveness of this process on enteric viruses. In this study, we compared the survival of two human norovirus surrogates, the feline calicivirus (FCV), and the murine norovirus (MNV-1), and of HAV on lettuce after water washing with bubbles and with or without ultrasound, and washing with bubbles in the presence of active chlorine (15 ppm) or peroxyacetic acid-based disinfectant (100 ppm). Cell culture and quantitative RT-PCR assays were used to detect and quantify the viruses on the surface of the lettuce after the sanitizing treatments. Levels of viral inactivation on the lettuce leaves were not significantly different between washing with bubbles and washing with bubbles plus ultrasound and were not dependant on the quantification method. A simple washing without disinfectant resulted in a decrease of approximately 0.7 log units in the quantity of virus detected for HAV and FCV and of 1.0 log unit for MNV-1.In the experimental set-up including a washing step (with or without ultrasound) followed by washing for 2 min in the presence of disinfectants, 15 ppm of active chlorine was found more effective for inactivating FCV (2.9 log units) than HAV and MNV-1 (1.9 log units and 1.4 log units, respectively) whereas 100 ppm of peroxyacetic-based biocide was found effective for inactivating FCV (3.2 log units) and MNV-1 (2.3 log units), but not HAV (0.7 log units). Quantitative RT-PCR results indicated that the presence of viral RNA did not correlate with the presence of infectious viruses on disinfected lettuce, except for MNV-1 processed with chlorine (15 ppm). In comparison with water washing, a substantial additional decrease of genomic FCV titer (1.1 log units) but no significant reduction of the genomic titers of HAV and MNV-1 were found on lettuce treated with chlorine (15 ppm). No significant effect of the disinfection step of lettuce with peroxyacetic-based biocide (100 ppm peracetic acid) was found by qRT-PCR on all genomic viral titers tested. This study illustrates the necessity of determining the effectiveness of technological processes against enteric viruses, using a relevant reference such as HAV, in order to reduce the risk of hepatitis and gastroenteritis by exposure to vegetables.     

诺如病毒检验用大肠杆菌噬菌体MS2标准物质的研制

目的 制备均匀性和稳定性符合要求的大肠杆菌噬菌体MS2标准物质,用于诺如病毒检验过程质量控制.方法 对大肠杆菌噬菌体MS2(CMCCPh001)进行分子生物学确认.采用冷冻干燥技术制成微球状标准物质,并进行均匀性、长期稳定性、短期稳定性检验.以牡蛎等5种样品作为基质,对使用效果进行确认.结果 制备的样品呈球形、白色、大...     

数字聚合酶链式反应法检测贝类和浆果中甲肝病毒

目的建立一种数字聚合酶链式反应(PCR)检测贝类和浆果中甲肝病毒的方法。方法样品经蛋白酶K消化-聚乙二醇法进行甲肝病毒富集后,使用高纯度病毒核酸试剂盒进行RNA提取,之后对甲肝病毒进行数字PCR检测。结果本方法对甲肝病毒有典型扩增,重复性和稳定性良好,对于草莓样品中甲肝病毒的检测灵敏度为25.30 CCID_(50)/20 g,树莓样品中甲肝病毒的检测灵敏度为6.32 CCID_(50)/20 g,贝类样品中甲肝病毒的检测灵敏度为12.54 CCID_(50)/2 g,表明其灵敏度高。结论该方法快速、准确、灵敏,适合测定贝类和浆果食品中甲肝病毒。     

Evaluation of different RNA-extraction kits for sensitive detection of hepatitis A virus in strawberry samples

The efficiency of different commercial RNA extraction kits for the detection of hepatitis A virus (HAV) from seeded strawberry samples was assessed by standard RT-PCR and real time RT-PCR (RT-qPCR). The best results with standard RT-PCR were achieved with Aurum™ Total RNA extraction kit (BioRad), obtaining a detection limit of 5-6.25 pfu/mg of tissue. A slightly lower sensitivity was rendered by the RNeasy^® Plant mini kit (Qiagen) (10-12.5 pfu/mg tissue), while the Total Quick RNA Cells and Tissues kit version mini (Talent) rendered a detection limit of 50-100 pfu/mg of tissue. The other tested commercial kits showed worse detection limits (>500 pfu/mg). With RT-qPCR and ten fold diluted RNA all the kits showed an increase of sensitivity, detecting the kits from Qiagen, Talent and BioRad down to 0.05 pfu/mg of strawberry homogenate. These findings indicate that the use of Aurum™ Total RNA extraction kit, with standard RT-PCR technique or RT-qPCR, can not only be labor and time saving but also helpful to improve the sensitivity for the HAV detection from fruits and to facilitate the standardization of detection methods among laboratories.     

Thermal inactivation of hepatitis A virus in suspension and in dried mussels (Mytilus edulis)

We examined the effects of three different temperatures (60, 85 and 100 °C) and durations of time (1 min at 100  ° C to 30 min at 60  ° C) on hepatitis A virus (HAV) in suspension and dried mussels (Mytilus edulis). In suspension, 3.61, 4.48, 5.06 and 5.66 log₁₀ tissue culture infectious dose (TCID)₅₀/mL were reduced by 60  ° C for 5 min, 60  ° C for 15 min, 60  ° C for 30 min and 85  ° C for 3 min, respectively. In dried mussels, 1.34, 1.94, 3.16, 2.36, 3.53 and 4.38 log₁₀TCID₅₀/mL were reduced by 60  ° C for 5 min, 60  ° C for 15 min, 60  ° C for 30 min, 85  ° C for 3 min, 85  ° C for 6 min and 85  ° C for 10 min, respectively. HAV inactivation from suspension and dried mussels was achieved by 85  ° C for 6 min and 15 min, respectively, and also by a 1 min at 100  ° C. At 60, 85 and 100  ° C, the 1‐log (D‐values) inactivation from both suspension and dried mussels was 6.33 and 7.93, 0.98 and 3.05, and 0.28 and 0.38 min, respectively. A higher temperature and/or a thermal treatment time shorter than 85  ° C for 6 min (100  ° C for 1 min) could be used for commercial target foods for complete HAV inactivation.     

贝类中甲型肝炎病毒磁珠免疫沉淀提取方法的建立及其与国标方法的比对

目的 建立蛋白酶K结合磁珠免疫沉淀提取贝类中甲型肝炎病毒(hepatitis A virus,HAV)的方法,并与国际标准方法(ISO15216—2017)进行比较研究.方法 应用高效价的HAV单克隆抗体,采用偶联技术,通过优化参数,建立蛋白酶K结合磁珠免疫沉淀的提取方法;通过人工预加入103～105 CCID50/m...     

Display of the antigenic region of Nipah virus nucleocapsid protein on hepatitis B virus capsid

The C-terminal domain of Nipah virus (NiV) nucleocapsid protein (NP_401–532) was inserted at the N-terminus and the immunodominant loop of hepatitis B core antigen (HBc). The stability of NP_401–532 increased tremendously when displayed on the HBc particles. These particles reacted specifically with the swine anti-NiV and the human anti-HBc antisera.     

Removal of Escherichia coli, Enterococcus fecalis, coliphage MS2, poliovirus, and hepatitis A virus from oysters (Crassostrea virginica) and hard shell clams (Mercinaria mercinaria) by depuration

Filter-feeding bivalve mollusks (shellfish) can bioaccumulate pathogenic microorganisms in up to 1000-fold higher levels than overlying waters, and therefore disease risks are associated with consuming raw or partially cooked shellfish. Many of these shellfish-borne diseases are due to enteric bacteria and viruses associated with fecal contamination. To control shellfish-borne diseases, guidelines for shellfish harvest waters and shellfish meat have been devised, which include cleansing of contaminated shellfish by depuration in controlled systems, heat pasteurization, or relay to clean waters. This study examines the depuration of oysters (Crassostrea virginica) and hard shell clams (Mercinaria mercinaria) in a flow-through depuration system under variable temperature (12 °C, 18 °C, and 25 °C), salinity (8 ppt, 18 ppt, and 28 ppt), turbidity (< 1 NTU, 10 NTU, and 20 NTU), pH (pH 7 and pH 8), and algae conditions (0 cells/mL and 50,000 cells/mL), with constant dissolved oxygen (5-7 mg/L). Oysters and hard shell clams were artificially contaminated with enteric microorganisms: Escherichia coli, Enterococcus faecalis, coliphage MS2, Poliovirus type-1 and Hepatitis A virus HM-175 (HAV), then depurated in 5-day trials with daily sampling. In oysters, optimizing environmental parameters of water temperature improved E. coli, MS2, poliovirus and HAV depuration, and optimized salinity improved E. coli, E. faecalis, and MS2 depuration rates. In hard shell clams, salinity improved E. coli and E. faecalis depuration rates. Adjusting turbidity, pH or algae did not improve microorganism depuration in either oysters or hard shell clams, with the exception of turbidity on E. faecalis in hard shell clams. Microorganism depuration rates in oysters from greatest to least were: MS2 > E. coli > E. faecalis > poliovirus > HAV, and in clams depuration rates from greatest to least were: E. coli > E. faecalis > HAV > MS2 > poliovirus. Because E. coli and E. faecalis were removed at faster rates than HAV and poliovirus, these fecal bacteria appear to be poor process indicators of the virological quality of depurated oysters and hard shell clams.     

贝类中甲肝病毒提取方法的比较

目的对贝类甲肝病毒的3种试剂盒提取方法进行比较。方法在贝类样品中添加不同滴度的甲肝减毒疫苗后,采用3种核酸提取法:Roche法、AM1836法、Qiagen74104法。按照其说明书提取模拟样品病毒核酸模板,从抽提效率、抽提核酸的稳定性、抑制剂去除效率3个方面对提取效果进行比较。结果在抽提核酸稳定性、抑制剂去除效率方面,3种方法结果相同,稳定性的Ct值为24.795～28.651,抑制剂去除效率方面提取的核酸10倍稀释核酸Ct值比未稀释核酸Ct值差值均为2.9～3.2左右(F=26.802,P0.05);在提取效率方面,Roche法高于AM1836法和Qiagen74104法,Ct值分别为28.5,28.7和29.0。结论 3种方法中,Roche法在贝类中甲肝病毒提取方面更有优势,可以提高贝类中甲肝病毒检测的灵敏度。     

Spectral sensitivity of Bacillus subtilis spores and MS2 coliphage for validation testing of ultraviolet reactors for water disinfection

The microbicidal UV fluence under polychromatic radiation from UV lamps is typically measured using the DNA absorbance spectrum as a weighting factor for the relative wavelength effectiveness. However, this DNA-based weighting does not necessarily match the spectral sensitivity of the microorganism being tested. Bacillus subtilis spores are often used for UV reactor validation in Europe, while MS2 coliphage is typically used for validation testing in the United States. These organisms were exposed to quasi-monochromatic UV irradiation across the microbicidal spectrum at wavelengths of 214, 230, 240, 254, 265, 280, and 293 nm. MS2 was three times more sensitive to wavelengths near 214 nm compared to the 254 nm output of low-pressure lamps, while B. subtilis spores were most sensitive to wavelengths around 265 nm. Use of these action spectra, compared to the DNA-based weighting, resulted in differences in the calculated polychromatic UV fluence. Consequently, the action spectrum, which is specific for each microorganism, has implications on the uncertainty of UV fluence determination during validation of reactors with polychromatic UV lamps.     

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。     

A predictive microbiology approach for thermal inactivation of Hepatitis A virus in acidified berries

Hepatitis A virus (HAV) is a food-borne enteric virus responsible for outbreaks of hepatitis associated with consumption of raw vegetables. Soft fruits, such as red berries, exposed to faecal contamination are increasingly responsible for collective food-borne illnesses associated with HAV, when eaten raw or used in unprocessed foods. Heat is the most effective measure for the inactivation of HAV. Thermal treatments are used on fruits as a decontamination method, but they have to be adapted to product characteristics; indeed, factors such as sugar or pH may have an impact on the viral sensitivity to thermal treatments. A model was developed for the inactivation of HAV in red berries without supplemented sugar and with different pH values. Nonlinear inactivation curves in acidified raspberries were modelled using an integrated model, with a single equation nesting secondary models of temperature and pH in the primary model. Model predictions were then confronted to experimental results obtained in another laboratory on other berries with different pH values. Excellent predictions were obtained in most cases, while failed predictions provided safe results, with the model predicting higher residual virus titres than what was observed.     

2014~2016年景洪市食品、公共场所 从业人员甲、戊肝调查分析

目的 分析景洪市2014~2016年食品、公共场所从业人员健康体检中甲肝、戊肝的结果, 为食品、公共场所从业人员健康体检工作的可持续发展提供数据支持。方法 对景洪市2014~2016食品、公共场所从业人员健康体检中戊型肝炎、甲型肝炎的检出率进行分析, 采用SPSS17.0进行?2检验。结果 2014~2016年食品、公共场所从业人员健康体检共检15897人, 总合格率99%。景洪市食品、公共场所从业人员甲型肝炎、戊型肝炎总体发病率呈下降趋势。其中, 甲型肝炎男性高发于女性; 戊型肝炎女性高发于男性; 甲型肝炎在公共场所行业高发于食品行业; 戊型肝炎阳性率无差异。结论 本市对于食品、公共场所从业人员的监管力度加强, 能够对感染源形成有效控制。     

戊型肝炎病毒食源性传播研究进展

戊型肝炎病毒（HEV）是一种全球性食源性病原体,猪、牛、兔、骆驼等多种动物均可成为其宿主。人类直接接触受HEV感染的猪和其他动物,或食用受污染的食物（未加工或未煮熟）均有感染该病毒的风险。本文综述了HEV的病原学、流行病学特征和在动物中的流行情况,以及对肉类及其制品、水生环境、水产品和蔬菜等作物的污染情况,可进一步了解HEV食源性传播风险,为预防和控制戊型肝炎的流行提供科学依据。