title: In silico and in vitro interrogation of a widely used HEV RT-qPCR assay for detection of the species Orthohepevirus A.
abstract: Hepatitis E virus (HEV) infection is a public health concern worldwide, associated with waterborne outbreaks in developing countries and reported as an emerging zoonotic infection in high-income countries. A recent consensus proposal classified the isolates from human, swine, wild boar, deer, mongoose, rabbit and camel in seven genotypes within the species Orthohepevirus A. In this report a popular HEV RT-qPCR assay was assessed for the detection of the species Orthohepevirus A. In silico analysis of 189 complete genome sequences showed that the assay targets a highly conserved region in the Orthohepevirus A genome. Additionally, plasmid standards were constructed to test the effect of probe- and primer-binding site mutations in the assay performance. The assay proved robust enough to detect strains with mutations in the probe-binding site and in the 3' end primer-binding site regions. A degenerate version of the reverse primer improves the performance of the assay particularly in the detection of HEV-5 and 6. The addition and detection of MS2 RNA in each RT-qPCR reaction monitored for amplification inhibition and did not affect the performance of the assay in the detection of the HEV RNA international standard. Therefore, the RT-qPCR assay can be confidently used for the RNA detection of the seven genotypes within the species Orthohepevirus A.
authors.affiliation: School of Life Sciences, University of Nottingham, Nottingham, UK. Electronic address: acarolinagcg@gmail.com.
authors.forename: Amalia
authors.initials: A
authors.lastname: Girón-Callejas
authors.affiliation: Department of Clinical Microbiology, Nottingham University Hospitals NHS Trust, Nottingham, UK. Electronic address: Gemma.Clark@nuh.nhs.uk.
authors.forename: Gemma
authors.initials: G
authors.lastname: Clark
authors.affiliation: School of Life Sciences, University of Nottingham, Nottingham, UK; Department of Clinical Microbiology, Nottingham University Hospitals NHS Trust, Nottingham, UK. Electronic address: will.irving@nottingham.ac.uk.
authors.forename: William L
authors.initials: WL
authors.lastname: Irving
authors.affiliation: School of Life Sciences, University of Nottingham, Nottingham, UK. Electronic address: patrick.mcclure@nottingham.ac.uk.
authors.forename: C Patrick
authors.initials: CP
authors.lastname: McClure
mesh_terms: D000818:Animals; D019295:Computational Biology; D016751:Hepatitis E / Q000175:diagnosis* / Q000821:virology; D006524:Hepatitis, Viral, Animal / Q000175:diagnosis* / Q000821:virology; D052678:Hepevirus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012327:RNA Virus Infections / Q000175:diagnosis* / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: HEV; Hepatitis E virus; Hepeviridae; Orthohepevirus A; Real-time PCR