Development of a novel recombinant encapsidated RNA particle: evaluation as an internal control for diagnostic RT-PCR.
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.
King
Donald P
DP
Institute for Animal Health, Ash Road, Pirbright, Surrey GU24 0NF, United Kingdom. donald.king@bbsrc.ac.uk
Montague
Nick
N
Ebert
Katja
K
Reid
Scott M
SM
Dukes
Juliet P
JP
Schädlich
Lysann
L
Belsham
Graham J
GJ
Lomonossoff
George P
GP
D000818:Animals; D017798:Comovirus / Q000235:genetics; D029822:Enterovirus B, Human / Q000302:isolation & purification*; D005536:Foot-and-Mouth Disease / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000302:isolation & purification*; D005822:Genetic Vectors; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine / Q000821:virology; D013555:Swine Vesicular Disease / Q000821:virology
C039919:RNA, recombinant; D012313:RNA