Development of a novel recombinant encapsidated RNA particle: evaluation as an internal control for diagnostic RT-PCR
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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.
|Journal||Journal of Virological Methods|
|Number of pages||8|
|Publication status||Published - Dec 2007|
- 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