Abstract: Objective To develop a multiplex real-time reverse transcription-PCR(RT-PCR) assay for rapid and simultaneous detection of norovirus genogroup I(GI) and genogroup II(GII) with Allglo probe.Methods Specific primers and Allglo probes were designed according to open reading frame 1(ORF1) and open reading frame 2(ORF2) target genes of norovirus GI and GII.The one-step real-time RT-PCR reaction system was established by optimizing reaction conditions.The specificity,anti-interference,and sensitivity of the multiplex real time RT-PCR assay were estimated and the assay was further validated with sequencing analyses of 96 clinical stool specimens.Results The results showed that the assay possessed high specificity for norovirus detection and without any evident cross-reaction with other viruses,including rotavirus,astrovirus and adenovirus.The detection limit of multiplex real-time RT-PCR assay for norovirus GI and GII were 10 copies /μL.Ninety-six clinical stool specimens were detected by both multiplex real-time RT-PCR assay and single real-time RT-PCR assay,with the matched results of 100%,and the results of sequencing analysis of these samples showed that the target sequence was correct.Conclusion The multiplex real-time RT-PCR assay by Allglo probe for the detection of norovirus GI and GII,with high specificity and sensitivity,was successfully established and the detection results of clinical specimems demonstrated that the method is useful for rapid screening of norovirus in outbreaks of acute gastroenteritis.