Rapid detection of Norovirus based on an automated extraction protocol and a real-time multiplexed single-step RT-PCR

J Clin Virol. 2006 Nov;37(3):156-61. doi: 10.1016/j.jcv.2006.06.014. Epub 2006 Sep 11.


Background: Molecular diagnosis of Norovirus infection can be a complex multistep process, which requires significant user intervention and expertise, and is not amenable to automation without extensive validation and optimization.

Objectives: To develop a real-time multiplexed RT-PCR assay with automated sample preparation that requires only a single-step and a single-tube for reverse transcription, amplification, and detection while exceeding the sensitivity of conventional PCR for broad-spectrum Norovirus detection.

Study design: Limit of detection was assessed against dilutions of clinical specimens. Fifty archived extractions were used to compare TaqMan sensitivity with either a separate RT using random primers or a single-step RT-PCR. The sensitivity of the novel assay was compared with conventional RT-PCR using 100 specimens from gastroenteritis cases.

Results: Automated extraction reduced RNA recovery by 0.5 logs compared to manual extraction but was more effective at removing PCR inhibitors from stool specimens. The optimized single-step real-time RT-PCR demonstrated no reduction in sensitivity. Together, the sensitivity of the novel assay was 19% higher than manual extraction with conventional RT-PCR.

Conclusions: A semi-automated and simplified molecular diagnostic protocol for the rapid detection of Norovirus has been achieved. PCR inhibitors are present in human fecal specimens and cause a significant problem for Norovirus detection by RT-PCR.

Publication types

  • Evaluation Study

MeSH terms

  • Caliciviridae Infections / diagnosis*
  • Child
  • Computer Systems
  • Feces / virology*
  • Humans
  • Molecular Diagnostic Techniques*
  • Norovirus / genetics
  • Norovirus / isolation & purification*
  • RNA, Viral / analysis*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction / standards


  • RNA, Viral