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

AbstractConventional 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. J. Med. Virol. 80:913–920, 2008. © 2008 Wiley‐Liss, Inc.