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

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.

Rapid detection of porcine encephalomyocarditis virus (EMCV) by isothermal reverse transcription recombinase polymerase amplification assays.

In this study, we combined reverse transcription recombinase polymerase amplification assay with the fluorescence detection platform (qRT-RPA) and lateral flow biosensor (LFB RT-RPA) to allow for rapid detection of porcine encephalomyocarditis virus (EMCV). Primers and probes were designed to target the highly conserved region of 3D gene of porcine EMCV. The optimal reaction condition of qRT-RPA and LFB RT-RPA was set as 42 °C for 20 min. The assays were highly specific to EMCV and no cross-reactions were observed with seven other porcine viruses. With a 10-fold serially diluted EMCV genomic RNA as template, the limit of detection was 1.0 × 102 and 1.0 × 101 copies for qRT-RPA assay and LFB RT-RPA assay, respectively. A total of 92 samples from different sources were examined using qRT-RPA, LFB RT-RPA and qRT-PCR. We found 100% diagnostic agreement between qRT-RPA (23/92) and qRT-PCR (23/92), and 97.83% diagnostic agreement between LFB RT-RPA (25/92) and qRT-PCR (23/92). There was no significant difference in performance between the RT-RPA assays developed in this study and a previously described qRT-PCR. However, RT-RPA assays were rapid and easy to perform while LFB RT-RPA exhibited higher sensitivity for EMCV than qRT-PCR. Therefore, the developed EMCV RT-RPA assays provide an attractive and promising tool for effective detection of EMCV in low-resource settings.