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

Food and Environmental Virology (Impact Factor: 2.36). 06/2011; 3(2):92-98. DOI: 10.1007/s12560-011-9062-9
Source: PubMed


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.

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    • "Required sample sizes were then calculated and delivered in chain-specific sampling plans to the data gathering laboratories. Samples were collected longitudinally per chain (Kokkinos et al., 2012; Maunula et al., 2013) and analyzed for norovirus (NoV; genogroups 1 and 2) and hepatitis A virus (HAV) with optimized, standardized detection procedures (D'Agostino et al., 2011; Martinez-Martinez et al., 2011). In addition to these human pathogenic viruses, the presence of human adenoviruses (hAdV) was examined to demonstrate that a route of contamination existed from infected humans to the sampling point, which other enteric viruses could follow. "
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