Absolute quantitation of RNA by a competitive real-time RT-PCR method using piscine nodavirus as a model.
The development and validation of a novel competitive real-time RT-PCR assay for the absolute quantitation of RNA from a piscine nodavirus are described. The assay utilises simultaneous amplification of target RNA and a recombinant RNA competitor in a single reaction, using the same pair of primers. The target and competitor products are distinguished by the use of two specific double-dye probes. The recombinant RNA competitor was designed to obtain a maximum sequence similarity with the target sequence, and the RT-PCR amplification efficiency of the competitor and target RNA was found to be identical. The intra-assay variation was 15-24% depending on the specific protocol for quantitation. The lower quantitation limit was estimated to 980 copies of RNA/reaction. The assay was used to evaluate the temporal development of the virus titre in an in vitro experiment, in which SSN-1 cell cultures were inoculated with nodavirus.
Grove
Søren
S
Department of Immunoprophylaxis, National Veterinary Institute, P.O. Box 8156 Dep., 0033 Oslo, Norway. soren.grove@vetinst.no
Faller
Randi
R
Soleim
Karen Baekken
KB
Dannevig
Birgit Helene
BH
D000704:Analysis of Variance; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D017931:DNA Primers; D005432:Flounder; D030381:Nodaviridae / Q000235:genetics* / Q000254:growth & development; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral; C039919:RNA, recombinant; D012313:RNA