Digital PCR for Quantifying Norovirus in Oysters Implicated in Outbreaks, France.
Ontology highlight
ABSTRACT:
SUBMITTER: Polo D
PROVIDER: S-EPMC5189157 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We investigated norovirus (NoV) concentrations and genotypes in oyster and faecal samples associated with two separate oyster-related outbreaks of gastroenteritis in Ireland. Quantitative analysis was performed using real-time quantitative reverse transcription polymerase chain reaction and phylogenetic analysis was conducted to establish the NoV genotypes present. For both outbreaks, the NoV concentration in oysters was >1000 genome copies/g digestive tissue and multiple genotypes were identified. In faecal samples, GII.13 was the only genotype detected for outbreak 1, whereas multiple genotypes were detected in outbreak 2 following the application of cloning procedures. While various genotypes were identified in oyster samples, not all were successful in causing infection in consumers. In outbreak 2 NoV GII.1 was identified in all four faecal samples analysed and NoV GII concentrations in faecal samples were >108 copies/g. This study demonstrates that a range of NoV genotypes can be present in highly contaminated oysters responsible for gastroenteritis outbreaks.
| S-EPMC9151252 | biostudies-literature
Project description:Noroviruses (NoVs) are the main agents of gastroenteritis in humans and the primary pathogens of shellfish-related outbreaks. Some NoV strains bind to shellfish tissues by using carbohydrate structures similar to their human ligands, leading to the hypothesis that such ligands may influence bioaccumulation. This study compares the bioaccumulation efficiencies and tissue distributions in oysters (Crassostrea gigas) of three strains from the two principal human norovirus genogroups. Clear differences between strains were observed. The GI.1 strain was the most efficiently concentrated strain. Bioaccumulation specifically occurred in digestive tissues in a dose-dependent manner, and its efficiency paralleled ligand expression, which was highest during the cold months. In comparison, the GII.4 strain was very poorly bioaccumulated and was recovered in almost all tissues without seasonal influence. The GII.3 strain presented an intermediate behavior, without seasonal effect and with less bioaccumulation efficiency than that of the GI.1 strain during the cold months. In addition, the GII.3 strain was transiently concentrated in gills and mantle before being almost specifically accumulated in digestive tissues. Carbohydrate ligand specificities of the strains at least partly explain the strain-dependent bioaccumulation characteristics. In particular, binding to the digestive-tube-specific ligand should contribute to bioaccumulation, whereas we hypothesize that binding to the sialic acid-containing ligand present in all tissues would contribute to retain virus particles in the gills or mantle and lead to rapid destruction.
| S-EPMC3126434 | biostudies-literature
Project description:Contaminated shellfish have been implicated in gastroenteritis outbreaks in different countries. As no regulation has been set up yet regarding viral contamination of food, very few data are available on the prevalence of contaminated products on the market. This study presents data obtained from oysters collected on the French market in one producing area over a 16 month period of time. Noroviruses were detected in 9% of samples with a seasonal impact and influence of climatic events. Contamination levels were low and, surprisingly, oysters sampled directly from the producer were found to have less contamination than oysters from supermarkets.
| S-EPMC3955953 | biostudies-literature
Project description:Human noroviruses (NoV) cause epidemics of acute gastroenteritis (AGE) worldwide and can be transmitted through consumption of contaminated foods. Fresh products such as shellfish can be contaminated by human sewage during production, which results in the presence of multiple virus strains, at very low concentrations. Here, we tested a targeted metagenomics approach by deep-sequencing PCR amplicons of the capsid (VP1) and polymerase (RdRp) viral genes, on a set of artificial samples and on shellfish samples associated to AGE outbreaks, to evaluate its advantages and limitations in the identification of strains from the NoV genogroup (G) II. Using artificial samples, the method allowed the sequencing of most strains, but not all, and displayed variability between replicates especially with lower viral concentrations. Using shellfish samples, targeted metagenomics was compared to Sanger-sequencing of cloned amplicons and was able to identify a higher diversity of NoV GII and GIV strains. It allowed phylogenetic analyses of VP1 sequences and the identification, in most samples, of GII.17[P17] strains, also identified in related clinical samples. Despite several limitations, combining RdRp- and VP1-targeted metagenomics is a sensitive approach allowing the study NoV diversity in low-contaminated foods and the identification of NoV strains implicated in outbreaks.
| S-EPMC7552006 | biostudies-literature
Project description:Oysters are major transmission vectors of noroviruses (NoVs) in the environment. Outbreaks of NoVs are often associated with the consumption of NoV-contaminated oysters. Laboratory confirmation of suspected oyster samples is a critical step in the surveillance and control of NoVs. Because of non-specific amplification, false-positive results are frequently obtained by semi-nested RT-PCR with the presently widely used primer set (G2SKF/G2SKR). Here, a novel universal PCR primer set N (NG2OF/NG2OR) specific for genogroup II (GII) NoVs was designed based on all GII NoV sequences available in public databases. Specific products were obtained with the primer set N when the NoV-positive oysters, spiked with each of five representative genotypes of GII NoVs (GII.17, GII.13, GII.4, GII.3, and GII.12), were subjected to analyzing. No products were detected with the primer set N for the NoV-negative oysters, while the primer set C gave various non-specific bands. Twenty-three out of 156 fresh oyster samples were NoV-positive with both the primer set N and the classic primer set, while eight were NoV-positive solely with the primer set N. Compared with the classic primer set, the newly designed primer set N had a higher detection rate and improved specificity for GII NoVs in oyster samples. These results show that the novel PCR primer pair is specific and applicable for the detection of GII NoVs in oysters.
| S-EPMC8802746 | biostudies-literature
Project description:Sensitive detection of water- and foodborne enteric viruses is extremely relevant, especially due to the low concentrations in which they are found. Accurate and sensitive detection of Norovirus, the primary responsible for water- and foodborne outbreaks, is of particular importance. Quantification of Norovirus is commonly performed by quantitative RT-PCR (RT-qPCR). In recent years a new platform was developed, digital PCR, that quantifies without the need for a standard curve thus decreasing the errors associated with its utilization. The platform developed by LifeTechnologies, QuantStudio 3D Digital PCR is amongst the least studied digital platform and although it allows the direct detection of DNA targets it requires a two-step RT-PCR for the detection of RNA targets. In this work we developed a new protocol able to detect Norovirus using a one-step digital PCR reaction (RT-dPCR). The performance of the newly developed one-step digital PCR was compared to RT-qPCR for the detection of Norovirus genogroup I and genogroup II. The sensitivity of RT-dPCR was identical to that of RT-qPCR, and the quantitative data determined by both methods were not significantly different for most samples. This one-step absolute quantification approach is a useful tool to minimize the time spent currently using this particular platform to amplify viral RNA and to standardize quantification of enteric viruses in food and environmental samples. This study proved the usefulness of the newly developed RT-dPCR protocol for a sensitive and accurate detection of low-copy targets.
| S-EPMC5531372 | biostudies-literature
Project description:Clostridium perfringens is both an ubiquitous environmental bacterium and the fourth most common causative agent of foodborne outbreaks (FBOs) in France and Europe. These outbreaks are known to be caused by C. perfringens enterotoxin (CPE) encoded by the cpe gene. However, additional information on the toxin/virulence gene content of C. perfringens has become available in the last few years. Therefore, to understand the enteropathogenicity of this bacterium, we need to describe the toxin and virulence genes content of strains involved in FBOs. In this study, we used a new real-time PCR typing technique based on a comprehensive set of 17 genes encoding virulence factors. The analysis was performed on a collection of 141 strains involved in 42 FBOs in the Paris region. It was combined with whole genome sequence (WGS) phylogenomic reconstruction, based on the coregenome single nucleotide polymorphisms (SNPs) of 58 isolates, representatives of the identified virulence gene profiles. Two or three different virulence gene profiles were detected in 10 FBOs, demonstrating that C. perfringens FBOs may be associated with heterogeneous strains. cpe-positive strains were isolated in 23 outbreaks, confirming the prominent role of CPE in pathogenicity. However, while C. perfringens was the sole pathogen isolated from the incriminated food, the cpe gene was not detected in strains related to 13 outbreaks. This result indicates either that the standard method was not able to isolate cpe+ strains or that the cpe gene may not be the only determinant of the enterotoxigenic potential of C. perfringens strains. Using phylogenomic reconstruction, we identified two clades distinguishing chromosomal cpe-positive from cpe-negative and plasmid-borne cpe. Important epidemiological information was also garnered from this phylogenomic reconstruction that revealed unexpected links between different outbreaks associated with closely related strains (seven SNP differences) and having common virulence gene profiles. This study provides new insight into the characterization of foodborne C. perfringens and highlights the potential of WGS for the investigation of FBOs.
| S-EPMC6481350 | biostudies-literature
Project description:[This corrects the article DOI: 10.3389/fmicb.2019.00777.].
| S-EPMC6619451 | biostudies-literature
Project description:As part of an intensified monitoring program for foodborne disease outbreaks in Finland, waterborne outbreaks were investigated for viruses. The diagnostic procedure included analysis of patients' stool samples by electron microscopy and reverse transcription-polymerase chain reaction (RT-PCR) for noroviruses and astroviruses. When these test results were positive for a virus, the water sample was analyzed. Virus concentration was based on positively charged filters from 1-L samples. Of the total 41 waterborne outbreaks reported during the observation period (1998-2003), samples from 28 outbreaks were available for analysis. As judged by RT-PCR results from patient samples, noroviruses caused 18 outbreaks. In 10 outbreaks, the water sample also yielded a norovirus. In all but 1 instance, the amplicon sequence was identical to that recovered from the patients. The ubiquity of waterborne norovirus outbreaks calls for measures to monitor water for viruses.
| S-EPMC3367355 | biostudies-literature
Project description:During the period from January 2000 to August 2005 a total of 204 outbreaks of norovirus gastroenteritis were diagnosed at the Norwegian Institute of Public Health. A clear increase in the norovirus activity was seen in healthcare institutions during the winter seasons. Polymerase sequence analysis of norovirus strains from 122 outbreaks showed that 112 were caused by GII strains (91.8%). Two norovirus variants seen during the study period-GIIb and GII.4-were predominant between January 2000 and September 2002, whereas GII.4 was predominant from September 2002 onward. The highest norovirus activity was seen during the 2002-2003 and 2004-2005 seasons with the emergence of new GII.4 variants. This study describes the molecular epidemiology of norovirus strains circulating in Norway during the five previous seasons and compares four norovirus real-time reverse transcriptase PCR assays. A suitable assay for routine diagnostics is suggested.
| S-EPMC1594767 | biostudies-literature