Design and Application of Nucleic Acid Standards for Quantitative Detection of Enteric Viruses by Real-Time PCR.

Food and Environmental Virology (Impact Factor: 2.51). 06/2011; 3(2):92-98. DOI: 10.1007/s12560-011-9062-9
Source: PubMed

ABSTRACT Synthetic multiple-target RNA and DNA oligonucleotides were constructed for use as quantification standards for nucleic acid amplification assays for human norovirus genogroup I and II, hepatitis E virus, murine norovirus, human adenovirus, porcine adenovirus and bovine polyomavirus. This approach overcomes the problems related to the difficulty of obtaining practical quantities of viral RNA and DNA from these viruses. The quantification capacity of assays using the standards was excellent in each case (R(2) > 0.998 and PCR efficiency > 0.89). The copy numbers of the standards were equivalent to the genome equivalents of representative viruses (murine norovirus and human adenovirus), ensuring an accurate determination of virus presence. The availability of these standards should facilitate the implementation of nucleic acid amplification-based methods for quantitative virus detection.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Viral gastroenteritis causes significant mortality and morbidity worldwide. Identifying the etiology of viral gastroenteritis is a challenge as most enteric viruses (EVs) are non-culturable. This study is to develop an EV testing panel using real-time PCR (EVPrtPCR) to simultaneously detect rotavirus, norovirus, sapovirus, astrovirus, and enteric adenovirus in stool samples. EVPrtPCR using universal amplification conditions was run in a single instrument run. EVPrtPCR was used to test 2,486 sporadic gastroenteritis samples submitted for EV testing using electron microscopy (EM) between July 2008 and July 2009. Retesting spiked negative stool samples and Salmon DNA as internal control were used to evaluate inhibition. EVPrtPCR detected viruses in significantly more samples: 748 (34%) as compared to 94 (3.8%) by EM. EM did not detect any norovirus, sapovirus, and mixed infection, and detected only 39% of rotavirus and 38.2% of enteric adenovirus positive samples. Four samples that tested positive for rotavirus and two for adenovirus and for small-round-structured viruses by EM were negative by EVPrtPCR. Norovirus was the most common virus detected (17.6%) with 92.4% as genogroup II, followed by rotavirus (6.8%), sapovirus (4.2%), astrovirus (2.0%), and enteric adenovirus (1.4%) with 9% samples positive for mixed infection. Overall, EV identification followed a U-shaped age distribution; positive samples were more common in children ≤5 years old and adults >60 years old. Norovirus, sapovirus and astrovirus showed winter predominance and rotavirus peaked in the spring. No inhibition was observed. Molecular technology significantly enhanced the identification of EV causing sporadic gastroenteritis in Alberta. J. Med. Virol. 9999: 1-8, 2013. © 2013 Wiley Periodicals, Inc.
    Journal of Medical Virology 11/2013; · 2.37 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In recent years, numerous foodborne outbreaks due to consumption of berry fruit contaminated by human enteric viruses have been reported. This European multinational study investigated possible contamination routes by monitoring the entire food chain for a panel of human and animal enteric viruses. A total of 785 samples were collected throughout the food production chain of four European countries (Czech Republic, Finland, Poland and Serbia) during two growing seasons. Samples were taken during the production phase, the processing phase, and at point-of-sale. Samples included irrigation water, animal faeces, food handlers' hand swabs, swabs from toilets on farms, from conveyor belts at processing plants, and of raspberries or strawberries at points-of-sale; all were subjected to virus analysis. The samples were analysed by real-time (reverse transcription, RT)-PCR, primarily for human adenoviruses (hAdV) to demonstrate that a route of contamination existed from infected persons to the food supply chain. The analyses also included testing for the presence of selected human (norovirus, NoV GI, NoV GII and hepatitis A virus, HAV), animal (porcine adenovirus, pAdV and bovine polyomavirus, bPyV) and zoonotic (hepatitis E virus, HEV) viruses. At berry production, hAdV was found in 9.5%, 5.8% and 9.1% of samples of irrigation water, food handlers' hands and toilets, respectively. At the processing plants, hAdV was detected in one (2.0%) swab from a food handler's hand. At point-of-sale, the prevalence of hAdV in fresh raspberries, frozen raspberries and fresh strawberries, was 0.7%, 3.2% and 2.0%, respectively. Of the human pathogenic viruses, NoV GII was detected in two (3.6%) water samples at berry production, but no HAV was detected in any of the samples. HEV-contaminated frozen raspberries were found once (2.6%). Animal faecal contamination was evidenced by positive pAdV and bPyV assay results. At berry production, one water sample contained both viruses, and at point-of-sale 5.7% and 1.3% of fresh and frozen berries tested positive for pAdV. At berry production hAdV was found both in irrigation water and on food handler's hands, which indicated that these may be important vehicles by which human pathogenic viruses enter the berry fruit chain. Moreover, both zoonotic and animal enteric viruses could be detected on the end products. This study gives insight into viral sources and transmission routes and emphasizes the necessity for thorough compliance with good agricultural and hygienic practice at the farms to help protect the public from viral infections.
    International journal of food microbiology 09/2013; 167(2):177-185. · 3.01 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Multiple outbreaks of human norovirus (hNoV) have been associated with fresh produce, such as soft berries and lettuce. Even though food handlers are considered an important source for the introduction of hNoV into food chains, their contribution to public health risks associated with hNoV remains unknown. To assess to which extent food handlers contribute to the introduction and spread of hNoV in fresh produce chains quantitative virus transfer data are needed. We estimated transfer proportions of hNoV GI.4, GII.4, murine norovirus (MNV-1), a culturable surrogate of hNoV, and human adenovirus (hAdV-2), a human pathogen proposed as an indicator for human faecal pollution, between gloved fingertips and raspberries, strawberries, and lettuce, by quantitative RT-PCR and cell culture if applicable. Virus transfer proportions were corrected for virus-matrix specific recoveries, and variability and uncertainty of the parameters were estimated. Virus transfer from gloves to soft berries was generally lower as compared to lettuce, with mean transfer proportions ranging between 0.1 to 2.3% and 9 to 10% for infectious MNV-1 and hAdV-2, respectively. Transfer from produce to glove was mostly greater than transfer from glove to produce, adding to the likelihood of virus transfer due to cross contamination from contaminated produce via food handlers. HNoV GI.4 and hNoV GII.4 showed no significant difference between their mean transfer proportions. Using the estimated transfer proportions, we studied the impact of low and high transfer proportions on the public health risk, based on a scenario in which a food handler picked raspberries with contaminated fingertips. Given the made assumptions, we could show that for a pathogen as infectious as hNoV, low transfer proportions may pose a greater public health risk than high transfer proportions, due to a greater viral spread. We demonstrated the potential of food handlers in spreading hNoV in food chains, showing that prevention of virus contamination on food handlers' hands is crucial for food safety. Nevertheless, complete prevention of virus contamination on fresh produce cannot be achieved in reality, and reliable and effective intervention measures are consequently required. We estimated that, especially for low transfer proportions, a robust one log10-unit reduction of infectious hNoV on contaminated produce, and on food handlers' hands, could lower the public health risk substantially. Using the obtained data in quantitative risk assessment will aid in elucidating the contribution of food handlers in hNoV transmission.
    International journal of food microbiology 08/2013; 166(3):419-425. · 3.01 Impact Factor

Full-text (2 Sources)

Available from
May 21, 2014