Detection of group A rotavirus in oyster tissues by in situ capture RT-qPCR

Group A rotavirus (RVA) is the major foodborne pathogen that could be transferred via oysters. There are limitations of current methods for detecting rotavirus in oysters, such as the long duration of the traditional cell culture-based plaque assay, the false positive due to the free RNA using RT-PCR-based molecular methods, and the complex process of RNA extraction from the food matrix. Therefore, we developed a PGM-mediated in situ capture reverse transcription quantitative polymerase chain reaction (ISC-RT-qPCR) assay to investigate the encapsulated RVA in oysters. In parallel comparing tests for the cell-cultured viral samples, the ISC-RT-qPCR exhibited 10-fold greater sensitivity and better accuracy of distinguishing the encapsulated RVA than RTqPCR. Furthermore, the ISC-RT-qPCR demonstrated a higher detection rate than RT-qPCR when applied to detect RVA in the artificially contaminated oyster. In practical application, the detection rates of RVA in oysters collected from four different farms were respectively higher by ISC-RT-qPCR (37.9%, 28.6%, 27.3%, and 0.0%) than by RT-qPCR (0.0%, 13.8%, 4.5%, and 0.0%), making the ISC-RT-qPCR a better assay for disease monitoring and control. Overall, all results indicate that ISC-RT-qPCR is a promising method for detecting encapsulated RVA in oysters and simplifies the steps of virus concentration, virus extraction with higher sensitivity and accuracy than the conventional RT-qPCR assay.