Development of a novel recombinant encapsidated RNA particle: evaluation as an internal control for diagnostic RT-PCR

Donald P. King, Nick Montague, Katja Ebert, Scott M. Reid, Juliet P. Dukes, Lysann Schhdlich, Graham Belsham, George P. Lomonossoff

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    This report describes the generation of novel encapsidated RNA particles and their evaluation as in-tube internal controls in diagnostic real-time reverse-transcription PCR (rRT-PCR) assays for the detection of RNA viruses. A cassette containing sequences of 2 diagnostic primer sets for foot-and-mouth disease virus (FMDV) and a set for swine vesicular disease virus (SVDV) was engineered into a full-length cDNA clone containing the RNA-2 segment of Cowpea Mosaic Virus (CPMV). After co-inoculation with a plasmid that expressed CPMV RNA-1, recombinant virus particles were rescued from cowpea plants (Vigna unguiculata). RNA contained in these particles was amplified in diagnostic rRT-PCR assays used for detection of FMDV and SVDV. Amplification of these internal controls was used to confirm that rRT-PCR inhibitors were absent from clinical samples, thereby verifying negative assay results. The recombinant CPMVs did not reduce the analytical sensitivity of the rRT-PCRs when amplification of the insert was performed in the same tube as the diagnostic target. This system provides an attractive solution to the production of internal controls for rRT-PCR assays since CPMV grows to high yields in plants, the particles are thermostable, RNase resistant and simple purification of RNA-2 containing capsids yields a preparation which is non-infectious.
    Original languageEnglish
    JournalJournal of Virological Methods
    Volume146
    Issue number1-2
    Pages (from-to)218-225
    ISSN0166-0934
    DOIs
    Publication statusPublished - 2007

    Keywords

    • Reverse transcription polymerase chain reaction
    • Virology
    • Real time
    • Method
    • Capsid
    • Microbiology

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