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

BackgroundMolecular 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.ObjectivesTo 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 designLimit 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.ResultsAutomated 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.ConclusionsA 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.