Objective: To develop a rapid, specific and sensitive diagnostic method for quantification and typing of genogroup I and II norovirus in oyster shellfish and stool samples from patients who had eaten them.
Methods: Specific primers and probe, following large scale norovirus genome consensus analysis were designed and subsequently a TaqMan based Real-time PCR assay to detect both GI and GII were established.
Results: This method showed high specificity for norovirus nucleic acid detection, and no cross-reaction among norovirus GI and GII. The limit on detection of NV genomes was 10(2) copies/microl. A total of 90 oysters and 37 stool specimens with diarrhea were tested for norovirus by conventional reverse transcriptional PCR (RT-PCR) assay as well as the TaqMan Real-time PCR, respectively. The norovirus detection rate in oysters by TaqMan PCR was significantly higher than that by conventional RT-PCR, but no differences between the two PCR methods were found when detecting the stool samples. Reliability of the Real-time PCR for norovirus detection was further confirmed by DNA sequencing of the positive samples
Conclusion: This TaqMan Real-time PCR assay was proved to be a useful method for quantification and typing for norovirus in routine monitoring of both oyster shellfish and clinical samples. This method is recommended to be an effective diagnostic method for outbreak-associated gastroenteritis due to norovirus.