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Development and Evaluation of a Multiplexed Real-Time TaqMan RT-PCR Assay with a Sample Process Control for Detection of F-specific RNA Coliphage Genogroups I and IV

  • Tineke H. Jones
  • Alain Houde
  • Elyse Poitras
  • Pierre Ward
  • Michael W. Johns
Original Paper

Abstract

There are increasing concerns of zoonotic transmission of some animal enteric viruses, such as calicivirus, hepatitis E virus, and rotavirus, which are closely related to human pathogenic strains. Most enteric viruses are detected by molecular techniques because they cannot be cultured. Surrogates such as F-RNA coliphages are cultivable but few molecular methods exist. Individual real-time TaqMan RT-PCR assays for the replicase gene of F-RNA coliphage genogroups I and IV were developed and multiplexed with a real-time TaqMan RT-PCR assay for feline calicivirus as a sample process control for the simultaneous detection and enumeration of genogroup I and IV F-RNA coliphages. Genogroup IV were successfully detected with the multiplexed assay in 80% of fecal samples that contained F-RNA coliphage levels ≥3.2 log plaque forming units (pfu). F-RNA coliphage were at or below the limit of detection in most fecal samples when levels were ≤4 log pfu/g.

Keywords

F+ RNA bacteriophages Virus surrogate Real-time RT-PCR Feline calicivirus Sample process control 

Notes

Acknowledgments

The authors thank Danielle Leblanc at AAFC, Food Research and Development Centre, St-Hyacinthe, QC, Canada, for her valuable discussion and FCV stock production and titration. This research was supported by Agriculture and Agri-Food Canada Research Branch Peer Reviewed Research Projects 75 and 162.

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Copyright information

© Her Majesty the Queen in Right of Canada  2009

Authors and Affiliations

  • Tineke H. Jones
    • 1
  • Alain Houde
    • 2
  • Elyse Poitras
    • 2
  • Pierre Ward
    • 2
  • Michael W. Johns
    • 1
  1. 1.Agriculture and Agri-Food CanadaLacombe Research CentreLacombeCanada
  2. 2.Agriculture and Agri-Food CanadaFood Research and Development CentreSt-HyacintheCanada

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