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.