Validation of an optimised method for quantitative detection of hepatitis E virus in pork sausage

• Hepatitis E virus (HEV) is an emerging zoonosis that can be transmitted to humans through the consumption of raw or undercooked pork meat products. Several methods for detecting the virus in food have been described, but there are still few robust data on qualitative and quantitative performance characteristics. In this study, we developed an optimised workflow for quantitative detection of HEV in pork sausage based on a combination of previously existing protocols. The protocol uses sample disruption and phase separation with tri-reagent and 1-bromo-3-chloropropane, followed by RNA concentration with isopropanol precipitation. We validated the protocol for use on reverse transcription quantitative real-time PCR (RT-qPCR) and reverse transcription droplet digital (RT-ddPCR). The 95% limit of detection and limit of quantification was 200 copies/g for both RT-qPCR and RT-ddPCR. The RT-ddPCR technology has previously shown promise as a more precise alternative to RT-qPCR. However, we found no evidence for improved performance using RT-ddPCR instead of RT-qPCR in this method. Furthermore, we also evaluated different combinations of RNA concentration methods and PCR detection strategies. This showed that isopropanol precipitation of viral RNA was more than twice as efficient as magnetic silica bead-based extraction when an inhibitor tolerant RT-PCR detection strategy was used. In conclusion, we present an efficient and well-characterised method for quantitative detection of HEV in pork sausage. Such methods are valuable to provide high quality data for risk assessments and food monitoring.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Mikrobiologi inom det medicinska området (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Microbiology in the medical area (hsv//eng)

Nyckelord

HEV

real-time PCR

digital PCR

foodborne virus

validation

method characterisation

pig

wild boar

Medicinsk vetenskap

Medical Science

Publikations- och innehållstyp

vet (ämneskategori)

ovr (ämneskategori)