Detection and quantification of human and bovine noroviruses by a TaqMan RT-PCR assay with a control for inhibition

Mol Cell Probes. 2008 Aug;22(4):215-22. doi: 10.1016/j.mcp.2008.02.003. Epub 2008 Mar 5.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cattle
  • Feces / virology
  • Humans
  • Molecular Sequence Data
  • Norovirus / genetics*
  • Norovirus / isolation & purification*
  • Reference Standards
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sequence Alignment
  • Staphylococcus aureus / genetics
  • Taq Polymerase / metabolism
  • Transcription, Genetic / genetics

Substances

  • Taq Polymerase