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 milises 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-I cell cultures were inoculated with nodavirus. (c) 2005 Elsevier B.V. All rights reserved.