Sensitive and rapid detection of norovirus using duplex TaqMan reverse transcription-polymerase chain reaction.

Conventional reverse transcription-polymerase chain reaction (RT-PCR) to detect norovirus (NV) is a complex of multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the final diagnosis. A duplex TaqMan RT-PCR was developed to detect and classify genogroup (G) I and GII of NV. The primers and TaqMan probes for this assay were selected from the region of open reading frame (ORF) 1-ORF2 junction. A total of 796 stool specimens from 103 outbreaks of gastroenteritis, and a series of 46 stool specimens containing most NV genotypes was used for this study. For these specimens from 103 outbreaks, NV was detected and classified by the duplex TaqMan RT-PCR in 536 of the 541 specimens tested previously to be positive using the conventional RT-PCR. Two hundred fifty-one of the 255 specimens that were negative by the conventional RT-PCR were also negative by the TaqMan RT-PCR. No false positive result was observed for other enteric RNA viruses such as rotavirus and sapovirus. This is the first report on the development of a duplex TaqMan RT-PCR end-point assay for detection and differentiation of GI and GII of NV strains simultaneously followed by genotyping. These results suggest the practical application of this duplex TaqMan RT-PCR is useful for the detection of NV in clinical specimens.