Development of a viability PCR assay for the analysis of Hepatitis E virus in food matrices

Abstract

Background: Hepatitis E virus (HEV) infection is considered an emerging problem in developed countries, where foodborne transmission plays a key role. As for other viruses, HEV detection in food matrices is based on molecular methods (PCR) that cannot discriminate between infectious and non-infectious virus. Viability PCR (v-PCR) is based on the use of intercalating dyes that penetrate through damaged viral capsid and block PCR amplification of their genome, therefore differentiating damaged virions from intact viruses. Aim of this study was the development of a v-PCR protocol for HEV in food matrices. Methods: A v-PCR protocol was optimized using a standardized amount of in vitro synthesized HEV RNA and ethidium bromide monoazide (EMA). Different EMA treatment conditions (incubation time from 15 to 120 minutes and concentrations from 20 to 320 microM) were tested. Efficiency of the treatment in removing free RNA was assessed by real-time RT(q)PCR. Control reactions were included to assess inhibition effects on PCR detection. The optimized v-PCR protocol was applied on HEV deactivated by heating (90 °C for 2 minutes) and on a food matrix (bivalve shellfish) experimentally contaminated with both intact and thermally treated virus. Results: Results showed that a 30 minute treatment with an EMA concentration of 200 microM provided a reduction of free HEV RNA ≥98% with little or no inhibition in the subsequent PCR reactions. Results were confirmed in the assays on thermally treated virus, while a lower reduction (∼60%) was obtained in experimentally contaminated shellfish, possibly in relation to dispersion of EMA activity onto nucleic acids resulting from the food matrix. Conclusions: The developed EMA-PCR protocol allows a better estimation of intact HEV particles compared to the standard real-time PCR protocol and, following optimization, can be applied for the detection of viable HEV in food matrices of animal origin. Key messages: - The analysis of foodborne viruses relies on molecular methods that may overestimate risk as a result of detection of free nucleic acids or damaged viral particles. - Viabilty PCR is a promising approach to differentiate infectious and non-infectious viruses in order to improve the assessment of the risk associated to viral contamination in foods.