Rapid and direct detection of hepatitis E virus in raw pork livers by recombinase polymerase amplification assays

Abstract

Hepatitis E virus (HEV) is a zoonotic pathogen that causes global hepatitis E. Outbreaks of hepatitis E are directly linked to the consumption of pork liver products. Herein reverse transcription recombinase polymerase amplification assays targeting the ORF2 gene were developed for the rapid detection of HEV by integrating the fluorescence detection platform (qRT-RPA) and the visible lateral flow biosensor by naked eyes (LFB RT-RPA). The qRT-RPA assay effectively detected HEV RNA with a limit of detection (LOD) of 154 copies/μl (95%CI: 126–333 copies/µl) in Genie III at 41°C for 20 min. Besides this, the LFB RT-RPA detected the HEV RNA with a LOD of 24 copies/μl (95%CI: 20–57 copies/µl) in an incubator block at 41°C for 20 min. The developed RT-RPA assays also showed good specificity for HEV, with no cross-reactions with any of the other important swine pathogens examined in this work. The performance of the developed RT-RPA assays was validated on 14 HEV RNA-positive and 66 HEV RNA-negative raw pork liver samples identified by a previously described qRT-PCR. Consequently, 11 and 12 samples were HEV RNA-positive as detected by the qRT-RPA and the LFB RT-RPA, respectively. Compared to qRT-PCR, the qRT-RPA and LFB RT- RPA assays revealed a coincidence rate of 96.3 and 97.5% as well as a Kappa value of 0.858 and 0.908, respectively. These results ascertain that the developed RT-RPA assays are effective diagnostic tools for the point-of-care detection of HEV in resource-limited settings.

Point-of-Care Testing for Hepatitis Viruses: A Growing Need

“…Using nucleic acids extracted from pig bile or shellfish, RT-LAMP can detect approximately 10 HEV RNA copies per microliter of extract in 60 min using turbidity or colorimetric techniques for visual identification of DNA amplification [197,198]. An assay using RT-RPA with homogenized pork livers was found to have a limit of detection of 34 HEV RNA copies per microliter of extracted sample in only 20 min using either real-time fluorescence measurements or lateral flow test strips [199]. Each of these studies uses laboratorybased methods for sample homogenization and nucleic acid extraction due to the inherent complexity of the shellfish and liver sample types.…”