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

Abstract

Human noroviruses (HuNoVs) are the major foodborne pathogens that cause non-bacterial gastroenteritis globally. Conventional RT-qPCR is prone to counting free RNA in the sample, resulting in an inflated infectious virus titer. Porcine gastric mucin (PGM), broadly used as the capture unit of in situ capture RT-qPCR (ISC-RT-qPCR), can precisely capture intact norovirus without adsorbing free RNA. However, PGM is a complex mixture that is difficult to obtain, and the concentration of histo-blood group antigens in it is unclear, hampering its widespread use. This study introduced a newly discovered capture unit, recombinant oyster heat shock protein 70 (roHSP70), to replace PGM. The roHSP70-mediated ISC-RT-qPCR (roHSP-ISC-RT-qPCR) were developed and compared with the PGM-mediated ISC-RT-qPCR (PGM-ISC-RT-qPCR) and conventional column-based RNA extraction RT-qPCR (CBE-RT-qPCR). The results showed that roHSP70 can capture various genotypes of HuNoVs. The smaller concentration of roHSP70 (20.0 μg/mL) captured HuNoVs to a similar extent compared to PGM (1.0 mg/mL). In addition, roHSP-ISC-RT-qPCR can accurately detect the amount of authentic intact virus particles in heat-treated Tulane virus (TV) samples with an accuracy comparable to that of TCID50. Interestingly, roHSP-ISC-RT-qPCR gave lower titers than CBE-RT-qPCR when measuring cell-cultured TV with high titers. However, when measuring lower-titer TV in artificially contaminated oysters, roHSP-ISC-RT-qPCR instead gave significantly higher titers than CBE-RT-qPCR. Overall, roHSP-ISC-RT-qPCR had a similar excellent performance as the PGM-ISC-RT-qPCR in detecting intact norovirus in samples. Moreover, roHSP70 has broader application scenarios than PGM due to its single-identified component and easier accessibility.