Development of recombinant oyster heat shock protein 70 mediated in situ capture RT-qPCR to detect human norovirus and Tulane virus

Authors: LYU, CHENANG - Shanghai Jiaotong University; LUO, GUANGDA - Shanghai Jiaotong University; AN, RAN - Shanghai Jiaotong University; Tian, Peng; WANG, DAPENG - Shanghai Jiaotong University

Submitted to: Food Control
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/2/2023
Publication Date: 12/7/2023
Citation: Lyu, C., Luo, G., An, R., Tian, P., Wang, D. 2023. Development of recombinant oyster heat shock protein 70 mediated in situ capture RT-qPCR to detect human norovirus and Tulane virus. Food Control. 158. Article 110245. https://doi.org/10.1016/j.foodcont.2023.110245.
DOI: https://doi.org/10.1016/j.foodcont.2023.110245

Interpretive Summary: Human noroviruses are the major non-bacteria foodborne pathogens. There are limitations of current olecular biology-based methods for detecting infectious norovirus in food. The free viral RNA in samples could yield false positive results. In coloration with scientists at Shanghai JiaoTong University, we identified a novel norovirus-binding protein from oyster (oyster heat shock protein 70). The oHSP70 shows a stronger capture ability than the previous capture unit, porcine gastric mucin (PGM, including histo-blood group antigens). In this study, roHSP70 was used to capture intact viruses followed by amplification of viral genomic copies by RT-qPCR. The method could capture and detect different genotypes of HuNoVs. In parallel comparing tests for TV (culturable surrogate for HuNoV), the roHSP70-mediated ISC-RT-qPCR is comparable to that of tissue culture-based assay (TCID50). The detection limits of roHSP70 based and PGM-based ISC-RT-PCR were between 2 copies/mL and 60 copies/mL In the detection of artificially contaminated oyster samples with TV, roHSP70-mediated ISC-RT-qPCR has a significantly higher viral recovery rate compared with the standard method (ISO 15216-2:2019 ) which was commonly used for detection of viruses in seafood. Overall, all results indicated that roHSP70-mediated ISC-RT-qPCR is a reliable method to detect wide genotypes of encapsulated HuNoVs in oysters and simplify the steps of virus concentration and RNA extraction with higher accuracy and sensitivity than the conventional RT-qPCR assay.

Technical Abstract: Human noroviruses (HuNoVs) are the major foodborne pathogen that causes non-bacterial gastroenteritis globally. The molecular assay is the current major way to monitor the contamination of HuNoVs in high-risk foods such as oysters. However, there are limitations of current methods for detecting infectious norovirus in food, such as the false positive result due to the free RNA of the virus and the false negative result because of the low titer virus. A modified in situ capture RT-qPCR (ISC-RT-qPCR) assay was developed using the recombinant oyster heat shock protein 70 (roHSP 70) as the capture unit to address this need. oHSP70 shows a stronger capture ability than the previous capture unit, porcine gastric mucin (PGM, including histo-blood group antigens). In this study, roHSP70-mediated ISC-RT-qPCR shows its ability to capture and detect different genotypes of HuNoVs and their surrogate, Tulane virus (TV). In parallel comparing tests fosed assay (TCID50). The detection limits of roHSP70 based and PGM-based ISC-RT-PCR were between 2 copies/mL and 60 copies/mL In the detection of artificially contaminated oyster samples with TV, roHSP70-mediated ISC-RT-qPCR has a significantly higher viral recovery rate compared with the standard method (ISO 15216-2:2019 ) which was commonly used for detection of viruses in seafood. Overall, all results indicated that roHSP70-mediated ISC-RT-qPCR is a reliable method to detect wide genotypes of encapsulated HuNoVs in oysters and simplify the steps of virus concentration and RNA extraction with higher accuracy and sensitivity than the conventional RT-qPCR assay.