Redesigned duplex RT-qPCR for detection of GI and GII human noroviruses

Authors: LIU, DANLEI - Shanghai Jiaotong University; ZHANG, ZILEI - Shanghai Jiaotong University; WU, QINGPING - Guangdong University; Tian, Peng; GENG, HAORAN - Shanghai Jiaotong University; WANG, DAPENG - Shanghai Jiaotong University

Submitted to: Engineering
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/30/2019
Publication Date: 3/3/2020
Citation: Liu, D., Zhang, Z., Wu, Q., Tian, P., Geng, H., Wang, D. 2020. Redesigned duplex RT-qPCR for detection of GI and GII human noroviruses. Engineering. 6(4):442-448. https://doi.org/10.1016/j.eng.2019.08.018.
DOI: https://doi.org/10.1016/j.eng.2019.08.018

Interpretive Summary: Human noroviruses (HuNoVs) are a major cause of non-bacterial acute gastroenteritis outbreaks worldwide. It accounts for more than 60,000 of hospitalization, 400,000 emergency room visits, two million outpatient visits, and 20 million total illnesses annually. As lacking an easy and reliable in vitro cultivation method, many approaches have been conducted for the detection of HuNoVs, including the electron microscopy, Enzyme-linked immunosorbent assay and molecular approaches (RT-PCR and RT-qPCR). Among which, RT-qPCR has been widely used as a gold standard for the detection of HuNoVs for its high sensitivity and specificity. In previous reports, primers have been taken from the ORF1-ORF2 junction, the most conserved region in the NoV genome. Kageyama reported the 1st primer-probe sets for GI and GII in 2003. After that, several similar primer-probe sets for HuNoVs have been reported from 2004 to 2007. So far, these sets of primers and probes are widely used in detection of HuNoVs without update. Due to the RNA nature of noroviral genome, mutation in viral genome occurs frequently and results in potential mismatch between once-perfectly matched primer/probe and changed viral sequences. In addition, more sequence data is available now than 10 years ago when all these primer/probes for RT-qPCR developed. More and more genotypes have been discovered with the rapid development of sequence technology and many of them might not even match the primer/probe designed 10 years ago at first place. Therefore, it is necessary to re-desired primers/probes with updated sequence database for a better detection of current strains of HuNoV. In this study, available genomic sequences of GI and GII HuNoV inputted since 2010 were collected from the GenBank and analyzed. Primer/probe sets for GI and GII HuNoV were re-designed (ND-RT-qPCR), evaluated and compared with the prototype Kageyama primer/probe (Kageyama RT-qPCR) in a set of clinical samples with representative genotypes of HuNoVs. All 5 GI samples were positive measured by ND-RT-qPCR and only 2 samples were positive by Kageyama RT-qPCR. All 18 GII samples were positive measured by ND-RT-qPCR and 17 samples were positive by Kageyama RT-qPCR. The sensitivity reflected by Ct value was better in ND-RT-qPCR than that of in Kageyama RT-qPCR. Our data suggested that ND-RT-qPCR could be a good fit for detection of current strains of HuNoVs.

Technical Abstract: Human noroviruses (HuNoVs) are major foodborne pathogens for nonbacterial acute gastroenteritis worldwide. As tissue-culture system for HuNoV is not mature enough for detection of the virus in routing, the detection is mainly depended on molecular approaches such as RT-PCR and RT-qPCR. The widely used primers and probes for RT-qPCR were established in the early of 2000s. As HuNoVs are highly variant viruses, mutations in viral genome makes perfectly matched primers and/or probes from previous design working less efficiency over time. In this study, we re-designed a new duplex real-time quantitative RT-PCR (ND-RT-qPCR) for detection of HuNoVs GI and GII based upon analysis of viral sequences inputted after 2010 in the database. By using long transcribed viral RNAs, we demonstrated that the sensitivity of ND-RT-qPCR was as low as 1 genomic copy for both GI and GII HuNoV. The performance of ND-RT-qPCR was further evaluated by comparing with the commonly used Kageyama primer-probe sets for RT-qPCR (Kageyama RT-qPCR) in 23 HuNoVs positive clinical samples. All 5 GI samples were positive measured by ND-RT-qPCR and only 2 samples were positive by Kageyama RT-qPCR. All 18 GII samples were positive measured by ND-RT-qPCR and 17 samples were positive by Kageyama RT-qPCR. The sensitivity reflected by Ct value was better in ND-RT-qPCR than that of in Kageyama RT-qPCR. Our data suggested that ND-RT-qPCR could be a good fit for detection of current strains of HuNoVs.