Hepatitis e virus (HEV) detection and quantification by a real-time reverse transcription-PCR assay calibrated to the world health organization standard for HEV RNA

Jeffrey J. Germer, Irina Ankoudinova, Yevgeniy S. Belousov, Walt Mahoney, Chen Dong, Jihong Meng, Jayawant Mandrekar, Joseph D. Yao

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA.

Original languageEnglish (US)
Pages (from-to)1478-1487
Number of pages10
JournalJournal of Clinical Microbiology
Volume55
Issue number5
DOIs
StatePublished - May 1 2017

Fingerprint

Hepatitis Viruses
Reverse Transcription
RNA
Polymerase Chain Reaction
Open Reading Frames
Genotype
Hepatitis E virus
RNA Probes
Bovine Viral Diarrhea Viruses
Immunocompromised Host
Chronic Hepatitis
Viral Load
Nucleotides
Confidence Intervals

Keywords

  • Hepatitis E virus
  • HEV RNA
  • Quantification
  • RT-qPCR
  • Viral load

ASJC Scopus subject areas

  • Microbiology (medical)

Cite this

Hepatitis e virus (HEV) detection and quantification by a real-time reverse transcription-PCR assay calibrated to the world health organization standard for HEV RNA. / Germer, Jeffrey J.; Ankoudinova, Irina; Belousov, Yevgeniy S.; Mahoney, Walt; Dong, Chen; Meng, Jihong; Mandrekar, Jayawant; Yao, Joseph D.

In: Journal of Clinical Microbiology, Vol. 55, No. 5, 01.05.2017, p. 1478-1487.

Research output: Contribution to journalArticle

Germer, Jeffrey J. ; Ankoudinova, Irina ; Belousov, Yevgeniy S. ; Mahoney, Walt ; Dong, Chen ; Meng, Jihong ; Mandrekar, Jayawant ; Yao, Joseph D. / Hepatitis e virus (HEV) detection and quantification by a real-time reverse transcription-PCR assay calibrated to the world health organization standard for HEV RNA. In: Journal of Clinical Microbiology. 2017 ; Vol. 55, No. 5. pp. 1478-1487.
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