Minor groove binder modification of widely used TaqMan probe for hepatitis E virus reduces risk of false negative real-time PCR results

Jeremy A. Garson*, Ruth B. Ferns, Paul R. Grant, Samreen Ijaz, Eleni Nastouli, Renata Szypulska, Richard S. Tedder

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    114 Citations (Scopus)

    Abstract

    Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in many parts of the developing world. It is responsible for both sporadic infections and large scale epidemics and may be associated with significant mortality during pregnancy. Over the past two decades many serological and nucleic acid based diagnostic tests for HEV have been developed, including several reverse transcription real-time polymerase chain reaction assays (RT-qPCR). One of the most widely used of these RT-qPCRs is that developed by Jothikumar and colleagues (Journal of Virological Methods 2006, 131, 65-71). Whilst reviewing this assay we calculated the predicted melting temperature of its TaqMan probe and consequently synthesised a minor groove binder (MGB) version in order to increase its hybridisation stability. In this report the performance of the original unmodified probe is compared with that of the MGB-modified version. We demonstrate that the MGB-modified probe detected HEV RNA in plasma samples from six patients with serologically confirmed hepatitis E in whom the unmodified probe had failed to detect HEV RNA. Sequence analysis of the ORF3 segment targeted by the RT-qPCR was possible in 4 of the 6 patients and revealed an identical C. →. T single nucleotide mutation in the probe binding region in each case.

    Original languageEnglish
    Pages (from-to)157-160
    Number of pages4
    JournalJournal of Virological Methods
    Volume186
    Issue number1-2
    DOIs
    Publication statusPublished - 2012

    Bibliographical note

    Funding Information:
    This work received a proportion of its funding from the Department of Health NIHR Biomedical Research Centres funding scheme.

    Keywords

    • HEV
    • Hepatitis E virus
    • Minor groove binder
    • Real-time PCR
    • TaqMan probe

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