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

Research output: Contribution to journalJournal articleResearchpeer-review

  • Donald P King
  • Nick Montague
  • Katja Ebert
  • Scott M Reid
  • Juliet P Dukes
  • Lysann Schädlich
  • Belsham, Graham John
  • George P Lomonossoff

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
Issue number1-2
Pages (from-to)218-25
Number of pages8
Publication statusPublished - Dec 2007

    Research areas

  • Animals, Comovirus/genetics, Enterovirus B, Human/isolation & purification, Foot-and-Mouth Disease/virology, Foot-and-Mouth Disease Virus/isolation & purification, Genetic Vectors, RNA/genetics, Reverse Transcriptase Polymerase Chain Reaction/methods, Sensitivity and Specificity, Swine/virology, Swine Vesicular Disease/virology

ID: 257918608