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See detailQuality assurance in quantitative microbial risk assessment
Boone, Idesbald; Van der Stede, Yves; Aerts, Marc et al

in Vlaams Diergeneeskundig Tijdschrift (2010), 79

Quantitative microbial risk assessment (QMRA) is being used to estimate the risk level of pathogens along the food chain and to support management decisions for the reduction of food-safety risks. The ... [more ▼]

Quantitative microbial risk assessment (QMRA) is being used to estimate the risk level of pathogens along the food chain and to support management decisions for the reduction of food-safety risks. The degree of credibility that can be attached to risk assessment results depends largely on the quality and quantity of the data, the model structure and the assumptions taken. Quality Assurance (QA) in QMRA is fulfilled when all steps in the QMRA process are technically and scientifically valid, so that it can meet its objectives. An overview of QA methods for QMRA is presented. Whereas sensitivity analysis and scenario analysis are common in QMRA, formal methods for the evaluation of data quality, the critical evaluation of assumptions, structured expert elicitation, the checklist approach, and peer review are rarely applied in QMRA but could largely improve the transparency in the results of QMRAs. The degree of implementation of these methods should be proportionate to the stakes of the risk management questions, and discussed in consultation between risk assessors and risk managers. [less ▲]

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See detailThe POLYGAL project : optimization of food conservation using a combination of lactates and polyphenols.
Dure, Rémi ULg; Ladeuze, S.; Martin, E. et al

Poster (2009, November)

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See detailQuantifying hand cross contamination in food.
Rodrigues, Ana; Dure, Rémi ULg; Delhalle, Laurent ULg et al

Poster (2009, June)

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See detailValidation of methods for the detection of new emerging pathogenic Escherichia coli
Verstraete, K; De Reu, K; Robyn, J et al

Book published by Brussels : Belgian Science Policy (2009)

Enterohaemorrhagic Escherichia coli (EHEC) are shigatoxin producing E. coli (STEC) that can cause serious disease to humans. These food-borne pathogens belong to the fifth most common zoonoses in Belgium ... [more ▼]

Enterohaemorrhagic Escherichia coli (EHEC) are shigatoxin producing E. coli (STEC) that can cause serious disease to humans. These food-borne pathogens belong to the fifth most common zoonoses in Belgium, but due to their severe clinical symptoms in humans they are highly dreaded. They can cause a range of disease symptoms ranging from asymptomatically carriage over various diarrhoea symptoms to the life-threatening HUS (haemolytic uremic syndrome). Cattle are the main reservoir and infection of humans occurs through contact with faecal excretion material and consumption of contaminated food or water. A broad variety of serotypes is able to cause human infections, but the principal serotypes are O26, O103, O111, O145 and O157. These strains are denoted as new emerging pathogens by the WHO. The group of sorbitol non-fermenting (s-) O157:H7 strains are examined the most, because an ISO-method is available. For sorbitol fermenting (s+) O157 strains as well as for non-O157 STEC strains recently a new isolation method was developed in the Belspo project SD/AF/06A (Possé et al. 2008a). The aim of the project was the optimization and the validation of the above-mentioned detection and isolation method for STEC in different matrices. In the first place immunomagnetic separation (IMS) was evaluated for the optimization of the STEC isolation method for cattle faeces (Ghent University, UGent). Second, molecular characterization of STEC strains was performed using a newly designed 33-mPCR as an alternative tool (University of Antwerp, VIB) and pulsed field gel electrophoresis (PFGE) (Institute for Agricultural and Fisheries Research, ILVO). Also a smaller derived multiplex PCR (9-mPCR) was designed (VIB) and optimized for the screening of samples (ILVO). The third goal was the evaluation of different approaches for STEC isolation from human faecal samples (Universitair Ziekenhuis Brussel, UZ). Finally the STEC detection and isolation method was validated by an in-house and an interlaboratory study which was based on the ISO 16140 guideline for the validation of alternative methods (University of Liège; UGent; ILVO). For the optimization of the STEC isolation protocol for cattle faeces and the evaluation of the effect of IMS, cattle faecal samples were artificially inoculated with various numbers of STEC (10-100 and 100-1000 cfu/25g faeces) and isolated using the isolation protocol with 6h or 24h of enrichment followed by IMS and plating or direct plating on selective agars. Two types of IMS beads (Dynabeads and Captivate beads) were tested. Results showed that IMS (any of the two types of beads) had a highly positive effect on the isolation of serotype O157 (s- and s+), whereas only a small or even a negative effect for non-O157 serotypes was found. This was largely clarified by results on pure broth suspensions of STEC, showing that high percentages were recovered from the IMS beads used in suspensions with the serotypes O157 (s- and s+), O26 and O103, but lower percentages were recovered for O111 and O145. Non-O157 STEC were often already efficiently isolated from faeces using only direct plating, whereas O157 (s- and s+) STEC were not. For the enrichment time, 24h generally gave higher isolation efficiencies than 6h. Finally for serotypes O157 (s- and s+), O26 and O103, a level of 10-100 cfu/25g was reliably detected, whereas for serotypes O111 and O145 only 100-1000 cfu/25g was reliably detected. To accomplish the second task of the project, the Applied Molecular Genomics Group of the VIB Department of Molecular Genetics (UA-VIB) designed a proprietary 33-amplicon multiplex PCR (mPCR) assay combined with capillary electrophoresis. This mPCR assay contains the detection of 5 STEC serotypes (O26, O103, O111, O145, O157), the main virulence genes VT1 with variants (VT1ab, VT1c and VT1d), VT2 with six variants (VT2b,c,d,e,f,g) and consensus, eae with five variants (eaeα1, eaeβ1, eaeγ1; eaeγ2; eaeε and eaeζ), ehx, tir, katP, saa, espP and FliC H2, H7, H8, H11 and H28. The assay was optimized and validated on a set of test strains representative for the priority amplicons. Next, this molecular technology was validated on a collection of 334 human clinical and animal strains from the Belgian STEC Reference Center (UZ). This collection of human and animal strains was also characterized by performing the PulseNet Europe protocol for pulsed field gel electrophoresis (PFGE). This technique creates a fingerprint of a strain by means of rare cutter restriction enzyme cutting of DNA and gel electrophoresis. Analysis of the band patterns lead to clustering of strains according to similarity or relatedness. Then results of 33-mPCR and PFGE genotyping were combined to show eventual correlations between PFGE genotypes and virulence profiles. Also background information about the strains (date of isolation, human or animal source, clinical manifestation, outbreak information) was included to the analysis. Combining mPCR and PFGE genotyping results, correlations were shown. In the first place STEC strains were clustered according to their serotype. Secondly a correlation occurred between virulence profile and PFGE clustering, concerning VT genes and other genes. Particularly for STEC O157, strains had very diverse VT-profiles, and strains with the same VT-profile clustered together. Concerning the clinical manifestation, ‘asymptomatic’ cases occurred more frequently for non-O157 than for O157 STEC, but besides this no correlation was shown between the PFGE clustering and the clinical manifestation or between the VT-profile and the clinical manifestation. Finally several case studies could be appointed based on the PFGE dendrograms. In general the cases contained clones that persisted during several years, had similar virulence profiles and infected humans as well as animals. As a part of the second task, the UA-VIB also designed a derived 9-amplicon multiplex PCR (9-mPCR) for fast sample screening. Using this 9-mPCR, a combination of serotypes (O26, O103, O111, O145, O157) and virulence genes (VT1, VT2, eae and ehx) is detected in one run and can be visualized using conventional gel electrophoresis. Once the 9-mPCR was developed and tested on pure strains, an evaluation on samples was performed. Hereto ILVO (Institute for Agricultural and Fisheries Research) tested several methods to extract DNA from artificially inoculated samples. Methods were compared based on the ability to remove PCR inhibiting molecules and on the ability to isolate and purify DNA from STEC cells. Out of four methods only two methods, in which no removal of sample debris was done, were suitable for sample preparation. The method using bead beating cell lysis described by Yu and Morrison (2004), was at least 10 times more sensitive than the method using the Qiagen Stool Mini Kit according to the manufacturer’s instructions, and was therefore recommended. However, the method using bead beating cell lysis is much more time consuming than the Qiagen method and the use of a ribolyser is necessary. As ILVO used the method employing the ribolyser in all following experiments, this method was used on artificially inoculated samples to determine its detection limit. All virulence marker genes and the serotype gene of strain MB3901 (serotype O157) could be detected in enriched minced beef and cheese from raw milk artificially inoculated with 2 cfu/25g sample. For cattle fecal samples the screening test was 10 times less sensitive; 21 cfu/25g feces could be detected. Finally the influence of the volume of lysate used in the mPCR reaction mix was examined. An mPCR reaction containing 1 and 2µl of lysate DNA was performed, but no difference in detection was seen. Testing of different clinical isolates of non-O157 STEC on the newly designed selective agars, showed that growth characteristics were generally as expected. However, more standardization of the preparation of the medium is needed to obtain more reproducible results. Some O103 isolates did not grow on the media prepared at UZ and the color of the colonies of O111 was often difficult to distinguish from O26. Using artificially contaminated stool samples, the sensitivity of the STEC isolation protocol developed in a previous Belspo SPSD II project was similar to the protocol used routinely at UZ (103 and 104 cfu/5g). The sensitivity was about 10 times higher when using IMS. The method performed well on frozen STEC positive samples, but this could only be tested on 14 samples, of which 11 with O157, 2 with O111 and one O26. In-house validation of the STEC isolation protocol was performed to evaluate if the protocol is applicable for different types of food matrices. All samples used for this validation were artificially contaminated. Ten samples of minced beef, raw milk cheese and sprouted seeds were artificially inoculated with varying numbers (10-2000 cfu/25g) of non-stressed and stressed strains belonging to the serotypes O157 (s-) and (s+), O26, O103, O111 and O145. Cultured STEC strains were cold and freeze stressed by storing them for at least 5 days at respectively 2 and -18°C. Inoculated samples were pre-enriched in a weak selective medium for 6 hours followed by enrichment in a stronger selective medium for 18 hours. Direct plating on a selective medium was performed after each enrichment step. In a third pathway, an IMS (Dynabeads or Captivate beads) step was performed after 24h enrichment and prior to plating. Suspected colonies on the selective medium were purified and tentatively confirmed on a purification medium followed by a confirmation by a serotype PCR. Parallel to the classical isolation method, the 9-mPCR screening test was performed on the enrichment medium (after 24 hours enrichment). Results indicate that the isolation protocol as well as mPCR screening provide good detection of non-stressed and cold-stressed O26, O103, O157 (s+) and O145 in raw milk cheese and minced beef. Detection of the other non-stressed and cold-stressed serotypes (O111 and O157 (s+)) in raw milk cheese and minced beef and of all serotypes under freeze stressed conditions in minced beef was low or almost zero. Probably due to the high level of background flora, detection of any serotype in sprouted seeds was almost impossible even though inoculation numbers were as high as 2000 cfu/25g. Finally the optimized STEC detection and isolation methods were validated by an interlaboratory study performed by national and international laboratories (twelve laboratories in total). First, a pre-trial experiment was organized to give the collaborative laboratories the possibility to become familiar with the isolation method. Secondly, the actual interlaboratory study was performed. Products necessary to prepare all culture media (in-house-prepared: IHP) and ready-to-use selective agar culture media (ready-to-use: RTU) were sent to the participating laboratories, as well as a questionnaire and a document to report the results. For each participating laboratory, 20 samples of 25g of minced beef were prepared: one sample for the temperature measurement upon arrival, one for the enumeration of the total count, Enterobacteriaceae and E. coli, two blank samples and sixteen samples inoculated with single strains belonging to 4 serotypes at 2 levels of contamination in duplicate (30 cfu/g and 300 cfu/g). All strains were cold stressed. Samples were prepared the day of the shipment and had to be analyzed on a prefixed day. The University of Liège evaluated all results based on the recommendations of ISO 16140. Results showed no difference between RTU and IHP media. The arabinose test seemed difficult to be read, so the dulcitol test is now preferred for the confirmation of serotypes O103 and O111. Some mistakes were made during sample inoculation, like a wrong inoculation of four samples and no inoculation of one sample. If we do not take into account these mistakes, all four serotypes were detected with high sensitivity. In general it can be concluded that the laboratory performance is highly satisfactory. [less ▲]

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See detailMETZOON : Development of a quantitative microbial risk assessment for human salmonellosis through household consumption of fresh minced pork méat in Belgium.
Bollaerts, Kaatje; Messens, Winy; Delhalle, Laurent ULg et al

in Risk Analysis : An Official Publication of the Society for Risk Analysis (2009), 29(6), 820-840

A quantitative microbial risk assessment according to the Codex Alimentarius Principles is conducted to evaluate the risk on human salmonellosis through household consumption of fresh minced pork meat in ... [more ▼]

A quantitative microbial risk assessment according to the Codex Alimentarius Principles is conducted to evaluate the risk on human salmonellosis through household consumption of fresh minced pork meat in Belgium. The quantitative exposure assessment is carried out by building a modular risk model, called the METZOON-model, which covers the pork production from farm to fork. In the METZOON-model, the food production pathway is split up in six consecutive modules: (1) primary production, (2) transport & lairage, (3) slaughterhouse, (4) post-processing, (5) distribution & storage and (6) preparation & consumption. All the modules are developed to resemble as closely as possible the Belgian situation making use of the available national data. Several statistical refinements and improved modeling techniques are proposed. The model produces highly realistic results. The baseline predicted number of annual salmonellosis cases is 20513 [st. dev. 9061.45]. The risk is estimated higher for the susceptible population [est. 4.713 × 10−5; st. dev. 1.466 × 10−5] compared to the normal population [est. 7.704 × 10−6; st. dev. 5.414 × 10−6] and is mainly due to cross contamination via cook’s hands and only for a small extent to undercooking. [less ▲]

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See detailNUSAP Method for Evaluating the Data Quality in a Quantitative Microbial Risk Assessment Model for Salmonella in the Pork Production Chain
Boone, Ides; Van der Stede, Yves; Bollaerts, Kaatje et al

in Risk Analysis : An Official Publication of the Society for Risk Analysis (2009), 29

The numeral unit spread assessment pedigree (NUSAP) system was implemented to evaluate the quality of input parameters in a quantitative microbial risk assessment (QMRA) model for Salmonella spp. in ... [more ▼]

The numeral unit spread assessment pedigree (NUSAP) system was implemented to evaluate the quality of input parameters in a quantitative microbial risk assessment (QMRA) model for Salmonella spp. in minced pork meat. The input parameters were grouped according to four successive exposure pathways: (1) primary production (2) transport, holding, and slaughterhouse, (3) postprocessing, distribution, and storage, and (4) preparation and consumption. An inventory of 101 potential input parameters was used for building the QMRA model. The characteristics of each parameter were defined using a standardized procedure to assess (1) the source of information, (2) the sampling methodology and sample size, and (3) the distributional properties of the estimate. Each parameter was scored by a panel of experts using a pedigree matrix containing four criteria (proxy, empirical basis, method, and validation) to assess the quality, and this was graphically represented by means of kite diagrams. The parameters obtained significantly lower scores for the validation criterion as compared with the other criteria. Overall strengths of parameters related to the primary production module were significantly stronger compared to the other modules (the transport, holding, and slaughterhouse module, the processing, distribution, and storage module, and the preparation and consumption module). The pedigree assessment contributed to select 20 parameters, which were subsequently introduced in the QMRA model. The NUSAP methodology and kite diagrams are objective tools to discuss and visualize the quality of the parameters in a structured way. These two tools can be used in the selection procedure of input parameters for a QMRA, and can lead to a more transparent quality assurance in the QMRA. [less ▲]

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See detailExpert Judgement in a risk assessment model for Salmonella spp. in pork : on the performance of different weighting schemes.
Boone, Ides; Van der Stede, Yves; Bollaerts, Kaatje et al

in Preventive Veterinary Medicine (2009), 92

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See detailSalmonella surveillance and control at post harvest in the Belgian pork meat chain.
Delhalle, Laurent ULg; Saegerman, Claude ULg; Farnir, Frédéric ULg et al

in Food Microbiology (2009), 26

Salmonella remains the primary cause of reported bacterial food borne disease outbreaks in Belgium. Pork and pork products are recognized as one of the major sources of human salmonellosis. In contrast ... [more ▼]

Salmonella remains the primary cause of reported bacterial food borne disease outbreaks in Belgium. Pork and pork products are recognized as one of the major sources of human salmonellosis. In contrast with the primary production and slaughterhouse phases of the pork meat production chain, only a few studies have focussed on the post-harvest stages. The goal of this study was to evaluate Salmonella and Escherichia coli contamination at the Belgian post-harvest stages. E. coli counts were estimated in order to evaluate the levels of faecal contamination. The results of bacteriological analysis from seven cutting plants, four meat-mincing plants and the four largest Belgian retailers were collected from official and self-monitoring controls. The prevalence of Salmonella in the cutting plants and meat-mincing plants ranged from 0% to 50%. The most frequently isolated serotype was Salmonella typhimurium. The prevalence in minced meat at retail level ranged from 0.3% to 4.3%. The levels of Salmonella contamination estimated from semi-quantitative analysis of data relating to carcasses, cuts of meat and minced meat were equal to 3.40 2.04 log CFU/cm2, 2.64 1.76 log CFU/g and 2.35 1.09 log CFU/g, respectively. The E. coli results in meat cuts and minced meat ranged from 0.21 0.50 to 1.23 0.89 log CFU/g and from 1.33 0.58 to 2.78 0.43 log CFU/g, respectively. The results showed that faecal contamination still needs to be reduced, especially in specific individual plants. [less ▲]

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See detailLes Salmonelles au niveau de la production primaire de porcs.
Korsak Koulagenko, Nicolas ULg; Delhalle, Laurent ULg; Daube, Georges ULg

Article for general public (2008)

La problématique des Salmonella spp. en filière animale, quelle que soit la spéculation envisagée, apparaît simple. De nombreux guides et ouvrages y ont été consacrés et, en théorie, il semble aisé de ... [more ▼]

La problématique des Salmonella spp. en filière animale, quelle que soit la spéculation envisagée, apparaît simple. De nombreux guides et ouvrages y ont été consacrés et, en théorie, il semble aisé de s’en débarrasser ou d’empêcher son introduction dans un élevage ou dans un atelier d’engraissement. En pratique, toutefois, la situation est différente et il est constaté que le micro-organisme peut contaminer la chaîne alimentaire en de multiples endroits, que ce soit au stade de la production primaire, dans le secteur abattage et transformation ou bien chez le consommateur. Pour arriver à développer une filière porcine « Salmonella-free », il faut une collaboration complète de tous les acteurs d’une filière, ce qui signifie qu’elle doit être intense et soutenue dans le temps. [less ▲]

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See detailSalmonella dans la filière porcine : de l'abattoir au consommateur
Delhalle, Laurent ULg; Korsak Koulagenko, Nicolas ULg; Daube, Georges ULg

Article for general public (2008)

Suite aux accords internationaux et à la modification de la législation européenne, l’analyse de risque est devenue une démarche systématique pour la maîtrise de la sécurité de la chaîne alimentaire ... [more ▼]

Suite aux accords internationaux et à la modification de la législation européenne, l’analyse de risque est devenue une démarche systématique pour la maîtrise de la sécurité de la chaîne alimentaire. L’analyse de risque permet de donner des réponses concrètes aux responsables de la santé, aux vétérinaires et aux industriels. Cette discipline permet de déterminer le risque pour une population donnée face à un danger, d’estimer le nombre de cas liés suite à l’ingestion d’une denrée contaminée, de simuler les conséquences d’un accident dans la chaîne alimentaire et de présenter les mesures de prévention efficaces. Elle permet également de proposer des scénarii possibles afin de réduire le nombre de cas et les coûts associés, d’évaluer l’effet de l’implémentation de mesures de gestion comme des critères de performance (par exemple des niveaux admissibles de contamination microbienne) ou des critères de traitement (par exemple une durée ou une température à une étape donnée), etc. L’analyse de risque peut être un guide quantitatif utile pour les prises de décision si elle s'appuie sur des études scientifiques solides et si elle est complétée par des décisions industrielles, sociales et politiques qui prennent en compte les limites de cette méthode. Actuellement, le Département des Sciences des Denrées alimentaires de la Faculté Vétérinaire de l’Université de Liège associés avec d’autres partenaires réalisent une évaluation quantitative de risque concernant Salmonella dans la filière porcine (projet METZOON). Le modèle développé simule la contamination par Salmonella de l’ensemble de la filière à partir de l’élevage de porc jusqu’à la consommation de la viande par la population. Le modèle fait appel aux résultats des plans de surveillance de l’AFSCA et à l’ensemble des études réalisées en Belgique sur les salmonelles dans la filière porcine. Plusieurs industriels ont également collaboré à cette étude. Le but est d’identifier les actions ayant le plus d’efficacité pour réduire la contamination de la chaîne alimentaire. En conclusion, le nombre de salmonelloses humaines a diminué ces dernières années en Belgique. L’implémentation des méthodes HACCP et des bonnes pratiques d’hygiène dans l’industrie, une meilleure surveillance des aliments par les autorités publiques, les campagnes de vaccination des poules pondeuses en production primaire sont les raisons essentielles de la diminution. Mais l’ensemble des acteurs de la filière doivent être conscients de leur responsabilité dans le but d’obtenir une viande indemne de salmonelles. Les bonnes pratiques de fabrication doivent être respectées tout au long de la chaîne de production, en accord avec les principes HACCP, avec des contrôles fréquents sur tous les points critiques. Une traçabilité complète de l’origine de la viande ou des produits de viande est nécessaire pour permettre aux autorités publiques et aux épidémiologistes d’établir l’origine d’épidémies et ainsi réagir au plus vite. Ce n’est qu’avec une collaboration continue de tous les acteurs que nous pourrons produire une viande sans salmonelles. [less ▲]

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See detailReducing human salmonellosis in Belgium from minced pork meat with quantitative risk assessment scenarios
Delhalle, Laurent ULg; Bollaerts, K.; Messens, W. et al

Poster (2008, May)

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See detailA structured expert judgement study on Salmonella spp. in pork: analyses of different weighting schemes.
Boone, Idesbald; Van der Stede, Y.; Bollaerts, K. et al

Poster (2008, March)

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See detailDetection and quantification of human and bovine novoviruses by a TaqMan RT-PCR assay with a control for inhibition.
Scipioni, Alexandra ULg; Bourgot, Isabelle; Mauroy, Axel ULg et al

in Molecular and Cellular Probes (2008), 22

Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and calves. Reverse transcription-polymerase ... [more ▼]

Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and calves. Reverse transcription-polymerase chain reaction (RT-PCR) has become the ‘‘gold standard’’ for detection of noroviruses in faecal and environmental samples. However, false negative results due to co-concentration of RT-PCR inhibitors are a continuous concern. A TaqMan real-time RT-PCR assay making use of a foreign internal RNA control and a RNA standard was developed. Very interestingly, this method is capable of detecting human noroviruses belonging to genogroups I and II, and bovine noroviruses belonging to genogroup III. Inhibitors were removed efficiently by 1/10 dilution of the sample or addition of bovine serum albumin to the RT-PCR mix. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. The ability to detect norovirus in stool samples that were negative by conventional RT-PCR assay demonstrate the higher sensitivity of the TaqMan assay compared to the conventional RT-PCR assay. This real-time RT-PCR assay allows the detection of both human and bovine noroviruses, avoids false negative results and is able to quantify the level of norovirus contamination. [less ▲]

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