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.     

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.     

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。     

Evaluation of an extracting method for the detection of Hepatitis A virus in shellfish by SYBR-Green real-time RT-PCR

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In the present study, we evaluated a rapid and simple extraction method to concentrate and purify enteric viruses from shellfish tissues for their detection by real-time RT-PCR. This procedure consists of an alkaline elution with a glycine buffer, solids removal by slow speed centrifugation, purification by chloroform extraction and virus concentration by ultracentrifugation. The efficiency of this method to recover Hepatitis A virus (HAV) from oysters seeded with this virus, was assessed by real-time RT-PCR and conventional RT-nested PCR after extracting viral RNA by a commercial isolation kit. Real-time RT-PCR yielded higher detection sensitivity than the obtained by conventional RT-nested PCR. Besides the improvements in detection sensitivity, the real-time RT-PCR, by quantifying HAV RNA, allowed to check the overall extraction procedure and the recovery efficiency after each processing step. After the last phase, i.e. virus concentration by ultracentrifugation, the RNA purity was high but the estimated HAV recovery efficiency was however low, probably due to virus losses and the presence of RT-PCR inhibitors in sample concentrates. In contrast, the HAV recovery percentage was higher after the virus elution step while the RNA purity was lower. Real-time RT-PCR detection could allow to eliminate some purification and concentration steps that are required for conventional RT-nested PCR detection. The overall procedure for detecting HAV could be then simplify avoiding virus losses during manipulation.     

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。     

三种分子生物学方法检测牡蛎中诺如病毒的比较研究

采用RT-PCR、RT- 半巢式PCR(seminested PCR)和RT- 环介导等温扩增(LAMP)三种分子生物学方法分别检测了牡蛎中经粪便污染的诺如病毒。三种方法所采用的特异性引物均针对诺如病毒高度保守的N/S 结构域。结果显示：一步法RT-PCR较两步法结果理想但仍不能有效去除食品中的PCR反应抑制物。RT- 半巢式PCR和RT-LAMP的特异性和敏感性都远远优于RT-PCR,但是RT- 半巢式PCR 操作繁琐费时。RT-LAMP 扩增程序简单、反应时间短,且不需要精密的温度循环装置,在产物中加入SYBR Green Ⅰ染料后可用肉眼直接判断反应结果。因此,RT-LAMP 有望发展成为快速检测牡蛎中诺如病毒的有效手段。     

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。     

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.     

季节性自回归差分移动平均模型在牡蛎中诺如病毒检出率预测上的应用

目的 基于季节性自回归差分移动平均(ARIMA)模型分析并预测上海市售牡蛎中诺如病毒(NoV)的检出率,为水产品中NoV的污染规律提供参考.方法 2016年6月-2019年11月,从上海芦潮港海鲜市场定期采购牡蛎样品共531只,通过巢式聚合酶链式反应(Nest-PCR),对其进行了 NoV检测,按季度分析检出率.采用季...     

Detection of hepatitis A virus in oysters

A digoxigenin-labelled RNA probe with a sensitivity of 800 50% tissue culture infectious dose (TCID₅₀) was used to detect Hepatitis A Virus (HAV) in oysters. We studied the influence of extraction methodology on riboprobe detection. Oyster samples obtained by four methods of extraction and extraction-concentration were spiked with HAV (CF53 strain). There was no correlation between protein concentration and turbidity of samples, and anti-digoxigenin antibodies showed a non specific reaction. Background noise was independent of protein concentration and disappeared when HAV RNA isolation by phenol/chloroform extraction was introduced, but HAV RNA could not be detected by this technique. In the presence of Acid Guanidinium Thiocyanate (AGT), RNA from HAV suspension was detected following phenolic extraction with a detection threshold of 8.10⁴ TCID₅₀ of spotted virus. HAV detection in oyster extract by a digoxigenin-labelled riboprobe appeared useful in shellfish virology, at least for a primary screening of samples.     

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.     

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。