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J Virol Methods. 2015 Mar;214:25-8. doi: 10.1016/j.jviromet.2014.11.025. Epub 2014 Dec 18.

In silico and in vitro interrogation of a widely used HEV RT-qPCR assay for detection of the species Orthohepevirus A.

Author information

1
School of Life Sciences, University of Nottingham, Nottingham, UK. Electronic address: acarolinagcg@gmail.com.
2
Department of Clinical Microbiology, Nottingham University Hospitals NHS Trust, Nottingham, UK. Electronic address: Gemma.Clark@nuh.nhs.uk.
3
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.
4
School of Life Sciences, University of Nottingham, Nottingham, UK. Electronic address: patrick.mcclure@nottingham.ac.uk.

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.

KEYWORDS:

HEV; Hepatitis E virus; Hepeviridae; Orthohepevirus A; Real-time PCR

PMID:
25528997
DOI:
10.1016/j.jviromet.2014.11.025
[Indexed for MEDLINE]

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