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Open Access Research

Detection of circulating norovirus genotypes: hitting a moving target

Brenda-Lee Rooney12, Janice Pettipas2, Elsie Grudeski3, Oksana Mykytczuk4, Xiao-Li Pang56, Tim F Booth3, Todd F Hatchette12 and Jason J LeBlanc12*

Author Affiliations

1 Dalhousie University, Halifax, Nova Scotia, Canada

2 Division of Microbiology, Department of Pathology and Laboratory Medicine, Capital District Health Authority (CDHA), Dalhousie University, Halifax, Nova Scotia, Canada

3 Enteroviruses and Enteric Viruses Laboratory, National Microbiology Laboratory (NML), Winnipeg, Manitoba, Canada

4 Food Virology Reference Centre, Bureau of Microbial Hazards, Health Canada, Ottawa, Ontario, Canada

5 Provincial Laboratory for Public Health (ProvLab), Edmonton, Alberta, Canada

6 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada

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Virology Journal 2014, 11:129  doi:10.1186/1743-422X-11-129

Published: 18 July 2014

Abstract

Background

Although national surveillance programs are in place to monitor norovirus epidemiology, the emergence of new strains and the genetic diversity among genotypes can be challenging for clinical laboratories. This study evaluated the analytical and clinical performance characteristics of one real-time RT-PCR and two end-point RT-PCRs commonly used in microbiology laboratories.

Methods

Lower limit of detection (LoD) was determined using 10-fold dilutions of noroviruses belonging to different genotypes. The clinical performance of the real-time and end-point RT-PCRs was assessed in parallel using nucleic acids extracted from 186 stool specimens.

Results

The real-time RT-PCR was highly sensitive and specific for the detection of norovirus genotypes that are currently circulating in Canada. In contrast, the two end-point RT-PCRs displayed poor analytical sensitivity or complete failure to detect certain norovirus genotypes, which was correlated to sequence mismatches in the primer-binding sites. In an attempt to improve norovirus detection with the end-point RT-PCRs, both assays were processed concurrently and detection from either assay was considered a positive result. Concurrent testing resulted in only a modest increase in clinical sensitivity (75.0%) compared to each assay alone (62.5% and 71.9%). However, the false positivity rate increased from 1.98% and 3.36% for the assays alone to 5.47% with concurrent testing.

Conclusions

This study emphasizes the benefits of a real-time method and provides support for routine surveillance to monitor norovirus epidemiology and ongoing proficiency testing to ensure detection of circulating norovirus genotypes.

Keywords:
Norovirus; Proficiency testing; Quantitative RT-PCR; Epidemiology; Genotyping