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Updated: Jul 20, 2026
Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR
Published on: July 22, 2012
Rapid detection of Norovirus based on an automated extraction protocol and a real-time multiplexed single-step RT-PCR
Nick A Antonishyn1, Nicole A Crozier, Ryan R McDonald
1Saskatchewan Health, Provincial Laboratory, Regina, Saskatchewan, S4S 5W6 Canada. nantonis@health.gov.sk.ca
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.
