کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
3408646 | 1593513 | 2006 | 9 صفحه PDF | دانلود رایگان |

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.
Journal: Journal of Virological Methods - Volume 132, Issues 1–2, March 2006, Pages 104–112