Development of a novel recombinant encapsidated RNA particle: evaluation as an internal control for diagnostic RT-PCR
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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.
Originalsprog | Engelsk |
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Tidsskrift | Journal of Virological Methods |
Vol/bind | 146 |
Udgave nummer | 1-2 |
Sider (fra-til) | 218-25 |
Antal sider | 8 |
ISSN | 0166-0934 |
DOI | |
Status | Udgivet - dec. 2007 |
ID: 257918608