Real-Time PCR-Based Methods for Detection of Hepatitis E Virus in Pork Products: A Critical Review.

Linda Scobie,Martin D’Agostino,Ann Wood,Nigel Cook

doi:10.3390/microorganisms10020428

Abstract

Standard methods for detection of hepatitis A virus and norovirus in at-risk foodstuffs are available, but currently there is no standard method for detection of hepatitis E virus (HEV) in pork products or other foods that can be contaminated with the virus. Detection assays for HEV are mainly based on nucleic acid amplification, particularly the reverse transcription polymerase chain reaction (RTPCR) in real-time format. RTPCR-based methods can be sensitive and specific, but they require a suite of controls to verify that they have performed correctly. There have been several RTPCR methods developed to detect HEV in pork products, varying in details of sample preparation and RTPCR target sequences. This review critically discusses published HEV detection methods, with emphasis on those that have been successfully used in subsequent studies and surveys. RTPCR assays have been used both qualitatively and quantitatively, although in the latter case the data acquired are only reliable if appropriate assay calibration has been performed. One particular RTPCR assay appears to be ideal for incorporation in a standard method, as it has been demonstrated to be highly specific and sensitive, and an appropriate control and calibration standard is available. The review focuses on the detection of HEV in pork products and similar foodstuffs (e.g., boar). The information may be useful to inform standardisation activities.

Highlights

The emergence of hepatitis E virus (HEV) as a zoonotic pathogen which may be transmitted by foods, especially through the pork supply chain, has resulted in a great deal of research being carried out by food and veterinary virologists across the globe to establish where links exist and how we may quantify the risks posed by the virus, and in developing methods to detect the virus in our foods [1]
This review focuses on the detection of HEV in pork products and similar foodstuffs using reverse transcription polymerase chain reaction (RTPCR) and the factors that need to be taken into consideration when evaluating detection data
HEV was detected in samples of pig liver sold at retail outlets in Germany by Wenzel et al [27], using the RTqPCR assay described by Jothikumar et al [20] using the human coxsackievirus B added to the sample prior to homogenisation as sample process control (SPC), but did not include an amplification control (AC) control

Summary

Introduction

The emergence of hepatitis E virus (HEV) as a zoonotic pathogen which may be transmitted by foods, especially through the pork supply chain, has resulted in a great deal of research being carried out by food and veterinary virologists across the globe to establish where links exist and how we may quantify the risks posed by the virus, and in developing methods to detect the virus in our foods [1]. The SPC is a non-target virus, added immediately upon sample receipt in the laboratory [11]; if it is detected with acceptable recovery efficiency the method is considered to have been effective for that sample Complex food matrices, such as pork products, can contain substances inhibitory to RTPCR, and if they are co-extracted with the target, reaction failure may result. Treatment of virus suspensions with RNase to destroy exposed RNA can selectively detect intact virus particles [14]; pre-treatment with RNA-intercalating dyes can prevent RTPCR from amplifying nucleic acid sequences from damaged viruses [15]. Neither of these adaptations, can selectively detect infectious virus.

Real-Time RTPCR-Based Detection Methods for HEV in Pork Products

Findings

Conclusions