Detection of hepatitis E virus genotype 3 in wastewater by an electrochemical genosensor

Abstract

Hepatitis E Virus (HEV) is an etiologic agent of hepatitis worldwide. HEV genotype 3 is the most prevalent in non-endemic regions, identified in humans, pigs and environmental samples. Thus, considering the zoonotic nature of HEV genotype 3, viral genome detection in wastewater concerns public health authorities. Electrochemical biosensors are promising analytical tools for viral genome detection in outside settings. This work reports on a highly specific, sensitive and portable electrochemical genosensor to detect HEV genotype 3 in wastewater samples. Based on the alignment analysis of HEV genotype 3 genome sequences available in GenBank, highly specific DNA target probes were designed to hybridize a target sequence within the ORF2/ORF3 overlapping genome region of HEV in between a biotinylated capture probe and a signal probe labeled with digoxigenin, in a sandwich-type format. An anti-Dig antibody labeled with the horseradish peroxidase (HRP) enzyme allowed electrochemical detection. The specificity of the target molecular probes of the viral genome was determined before the biosensor assembly by in silico analysis, PCR and qPCR assays demonstrating efficient amplification of two targets, i.e., nucleotides 5338–5373 and 5328–5373, but this last one of higher performance. The electrochemical response of the genosensor with synthetic HEV was target concentration-dependent in a linear range from 300 pM to 2.4 nM, with a sensitivity of 16.93 μA/nM, a LOD 1.2 pM and high reproducibility. The genosensor response was differential when interrogated with the HEV genotype 3 viral genomes from wastewater against other four viruses. Therefore, the approach offers a step forward to the epidemiologic surveillance of viruses in wastewater as an early warning system.