An internally controlled, one-step, real-time RT-PCR assay for norovirus detection and genogrouping

J Clin Virol. 2007 Aug;39(4):318-21. doi: 10.1016/j.jcv.2007.05.005. Epub 2007 Jun 28.

Abstract

Background: Reverse transcription (RT)-PCR for norovirus detection is prone to false-negative results due to inhibitory substances in faeces. An internal control is needed to monitor extraction efficiency and to detect inhibition.

Objectives: To further develop a one-step RT-PCR assay for norovirus detection/genogrouping by addition of MS2 bacteriophage as an internal control.

Study design: Our norovirus RT-PCR assay was modified by addition of MS2 phage to the extraction tray and primers/probe for MS2 detection to the reaction mix. The effect of addition of MS2 phage and MS2 primers/probe on the sensitivity/specificity of the PCR assay was examined.

Results: The addition of MS2 as an internal control showed no loss of sensitivity or specificity for norovirus detection.

Conclusions: A triplex, one-step, type-specific, real-time RT-PCR with MS2 internal control has been developed for use in routine laboratory diagnosis of norovirus infection.

Publication types

  • Evaluation Study

MeSH terms

  • Caliciviridae Infections / virology*
  • DNA Primers
  • Feces / virology
  • Gastroenteritis / virology*
  • Genotype
  • Humans
  • Levivirus / genetics
  • Levivirus / isolation & purification
  • Norovirus / classification*
  • Norovirus / genetics
  • Norovirus / isolation & purification*
  • RNA, Viral / analysis
  • RNA, Viral / isolation & purification
  • RNA, Viral / standards*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / standards
  • Sensitivity and Specificity

Substances

  • DNA Primers
  • RNA, Viral