Redesigned Duplex RT-qPCR for the Detection of GI and GII Human Noroviruses

Human noroviruses (HuNoVs) are major foodborne pathogens that cause nonbacterial acute gastroenteritis worldwide. As the tissue-culture system for HuNoVs is not mature enough for routine detection of the virus, detection is mainly dependent on molecular approaches such as reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The widely used primers and probes for RT-qPCR were established in the early 2000s. As HuNoVs are highly variant viruses, viral genome mutations result in previously designed primers and/or probes that were perfectly matched working less efficiently over time. In this study, a new duplex RT-qPCR (ND-RT-qPCR) was designed for the detection of genogroup I (GI) and genogroup II (GII) HuNoVs based on an analysis of viral sequences added in the database after 2010. Using long transcribed viral RNAs, the results demonstrate that the sensitivity of ND-RT-qPCR is as low as one genomic copy for both GI and GII HuNoVs. The performance of ND-RT-qPCR was further evaluated by a comparison with the commonly used Kageyama primer/probe sets for RT-qPCR (Kageyama RT-qPCR) for 23 HuNoV-positive clinical samples. All five GI samples were registered as positive by ND-RT-qPCR, whereas only two samples were registered as positive by Kageyama RT-qPCR. All 18 GII samples were registered as positive by ND-RT-qPCR, while 17 samples were registered as positive by Kageyama RT-qPCR. The sensitivity reflected by the quantification cycle (Cq) value was lower in ND-RT-qPCR than in Kageyama RT-qPCR. Our data suggest that ND-RT-qPCR could be a good fit for the detection of current strains of HuNoVs.