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。     

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...     

2015年甘肃省白银市食源性腹泻患者诺如病毒检测结果分析

目的 分析2015年甘肃省白银市食源性疾病监测人群诺如病毒检测结果,为科学制定诺如病毒引起食源性疾病的预测、预警及防控提供参考依据。方法 对在医院就诊的344例食源性疾病监测病例进行问卷调查,采集粪便标本,应用实时荧光定量聚合酶链式反应(荧光定量RT-PCR)方法检测诺如病毒GI和GII基因组。结果 2015年白银市报告的344例食源性疾病病例中,检出诺如病毒阳性病例78例,检出率为22.7%(78/344),其中诺如病毒GI基因组阳性5例,GII基因组阳性71例,GI/GII基因组混合感染2例;男性46例,女性32例;阳性患者中年龄最大者83岁,最小者仅3个月,平均年龄为20.3岁。结论 2015年白银市食源性疾病病例中,诺如病毒检出率较高,全年均流行,但流行季节主要在秋冬季,诺如病毒GII基因组为优势组,应加强公众科普宣传教育,提高诺如病毒监测检测能力,完善风险管理措施。     

一起疑似食源性诺如病毒胃肠炎事件病原分子生物学特征分析

目的研究厦门市思明区一起聚集性胃肠炎事件的诺如病毒的分子生物学特征。方法将收集到的11份肛拭子标本及1份生蚝样品,采用实时荧光定量逆转录-聚合酶链反应(RT-PCR)方法检测诺如病毒核酸,阳性标本再进行实时荧光定量RT-PCR扩增,扩增产物经过凝胶电泳分析后,进行序列测定,确定基因型,并进行系统发育树分析。结果 11份肛拭子标本中检出5株GⅠ组诺如病毒株,2株测序成功,检出4株GⅡ组毒株,3株测序成功;1份生蚝样品中检出1株GⅠ组毒株,测序成功。对测序成功的6株毒株进行同源性分析,GⅠ.2型毒株所测序列与2014年上海株KP325648等6株参考株高度同源,GⅡ.17型毒株所测序列与2015年韩国株KT384078等8株参考株高度同源,证实这是一起由GⅠ.2和GⅡ.17型诺如病毒混合感染引起的聚集性胃肠炎事件,GⅡ.17型毒株为厦门市首次报道。结论此次聚集性胃肠炎事件是由诺如病毒引起,且为GⅠ.2与GⅡ.17型毒株混合感染引起。     

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.     

秦皇岛市售生菜、草莓中诺如病毒监测分析

目的 调查秦皇岛市诺如病毒污染的高风险食品生菜和草莓中诺如病毒的污染状况。方法 2015年9月至2016年6月采集市售的生菜201份、草莓136份, 利用实时荧光RT-PCR法检测诺如病毒, 在样品中添加Mengo病毒进行过程控制, 并根据Mengo病毒标准曲线计算 诺如病毒回收率。结果 Mengo病毒回收率均达到了ISO/TS 15216-2-2013对 病毒回收率＞1%的要求。同时对201份生菜样品进行检测, 检出诺如病毒阳性标本9份, 总检出率为4.5%, 其中GⅠ型检出率为0.5%(1/201); GⅡ型检出率为3.5%(7/201); GⅠ型和GⅡ型同时检出率为0.5%(1/201)。136份草莓样品中检出阳性标本4份, 总检出率为2.9%, 其中GⅠ型检出率为0.7%(1/136); GⅡ型检出率为2.2%(3/136)。结论 秦皇岛市售蔬菜、浆果中部分存在诺如病毒污染, 诺如病毒检出率在不同采样环节以及时间分布比较差异无统计学意义, 需要进一步加强日常监测。     

Detection of noroviruses in shellfish in the Netherlands

Shellfish from oyster farms in the Netherlands and imported from other European countries were examined for viral contamination. A method that allows sequence matching between noroviruses from human cases and shellfish was used. The samples of shellfish (n = 42) were analyzed using a semi-nested RT-PCR that had been optimized for detection of norovirus in shellfish (SR primer sets). In addition, a different genome region was targeted using a second primer set which is routinely used for diagnosis of norovirus infection in humans (JV12Y/JV13I). To improve the detection limit for this RT-PCR a semi-nested test format was developed (NV primer sets). One of 21 oyster samples (4.8%) from Dutch farms was norovirus positive, whereas norovirus was detected in 1 out of 8 oyster samples (12.5%) and 5 out of 13 mussel samples (38.5%) collected directly after importation in the Netherlands. RNA from samples associated with an outbreak of gastro-enteritis in the Netherlands in 2001 was re-analyzed using the NV primer sets. At least one identical sequence (142/142 nt) was found in three fecal and in two oyster samples related to this outbreak. Further surveillance of norovirus by detection and typing of viruses from patients with gastroenteritis and shellfish is warranted to clarify the causes of future outbreaks.     

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.     

Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11

The presence of maize intrinsic and recombinant cry1Ab genes in the gastrointestinal (GI) contents, peripheral blood mononuclear cells (PBMC), and visceral organs of calves fed genetically modified Bt11 maize was examined by PCR in a subchronic 90-day performance study. Samples were collected from six Japanese Black/Holstein calves fed Bt11 maize and from six calves fed non-Bt maize. Fragments of maize zein (Ze1), invertase, chloroplast, and cry1Ab were detected inconsistently in the rumen fluid and rectal contents 5 and 18 h after feeding. The chloroplast DNA fragments of ribulose-1,5-bisphosphate carboxylase/oxygenase and tRNA were detected inconsistently in the PBMC, the visceral organs, and the longissimus muscle, while the cry1Ab gene was never detected in PBMC or in the visceral organs. These results suggest that feed-derived maize DNA was mostly degraded in the GI tract but that fragmented DNA was detectable in the GI contents as a possible source of transfer to calf tissues. These results also suggest that the recombinant cry1Ab genes were not transferred to the PBMC and tissues of calves fed Bt11 maize.     

Adsorption and aggregation properties of norovirus GI and GII virus-like particles demonstrate differing responses to solution chemistry

The transport properties (adsorption and aggregation behavior) of virus-like particles (VLPs) of two strains of norovirus ("Norwalk" GI.1 and "Houston" GII.4) were studied in a variety of solution chemistries. GI.1 and GII.4 VLPs were found to be stable against aggregation at pH 4.0-8.0. At pH 9.0, GI.1 VLPs rapidly disintegrated. The attachment efficiencies (α) of GI.1 and GII.4 VLPs to silica increased with increasing ionic strength in NaCl solutions at pH 8.0. The attachment efficiency of GI.1 VLPs decreased as pH was increased above the isoelectric point (pH 5.0), whereas at and below the isoelectric point, the attachment efficiency was erratic. Ca(2+) and Mg(2+) dramatically increased the attachment efficiencies of GI.1 and GII.4 VLPs, which may be due to specific interactions with the VLP capsids. Bicarbonate decreased attachment efficiencies for both GI.1 and GII.4 VLPs, whereas phosphate decreased the attachment efficiency of GI.1, while increasing GII.4 attachment efficiency. The observed differences in GI.1 and GII.4 VLP attachment efficiencies in response to solution chemistry may be attributed to differential responses of the unique arrangement of exposed amino acid residues on the capsid surface of each VLP strain.     

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。     

我国食源性诺如病毒GII.2[P2]型毒株基因组结构与衣壳蛋白功能研究

食源性诺如病毒（Norovirus）是引起全球食品安全事件的主要微生物病原,具有丰富的遗传多样性,基因型较复杂,近年来GII.2逐渐成为我国主要流行基因型之一。为了解食源性诺如病毒GII.2型变异特点,该研究以2015年在广州地区检出的GII.2[P2]型GZ2015-L335毒株为对象,对其基因组结构及衣壳蛋白功能进行了系统表征。该毒株基因全长为7 496 bp,在线基因分型结果表明其为GII.2[P2]基因型。通过克隆其衣壳蛋白VP1基因,借助杆状病毒表达系统获取重组VP1蛋白,并采用氯化铯密度梯度离心法进行纯化。SDS-PAGE和Western blot实验结果表明重组VP1蛋白分子量约为58 ku;透射电镜结果显示该蛋白可自组装形成直径约为30 nm的病毒样颗粒（Virus-like Particle,VLP）;ELISA结果显示该VLP与抗GII.2[P]衣壳P颗粒血清具有较高结合活性。此外,受体结合实验证实该VLP与A/B/O等分泌型及非分泌型唾液呈现出广泛的结合作用。该研究对GII.2[P2]型GZ2015-L335毒株基因组结构进行了系统分析,成功制备并表征了其病毒样颗粒,结果将为食源性诺如病毒的高效抗体研制及新型检测技术研发提供支撑。     

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.     

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。