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Title: A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2)
Authors: Duarte, Margarida Dias
Carvalho, Carina Luísa
Barros, Sílvia
Henriques, Ana M.
Ramos, Fernanda
Fagulha, Teresa
Luís, Tiago
Duarte, Elsa Leclerc
Fevereiro, Miguel
Keywords: Rabbit hemorrhagic disease virus
Taqman RT-PCR
Real time RT-qPCR
Issue Date: Jul-2015
Publisher: Elsevier
Citation: Duarte et al. (2015) A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2). J. Virol. Met. 219, July 2015, Pages 90–95.
Abstract: A specific real time RT-PCR for the detection of RHDV2 was developed and validated using RHDV and RHDV2 RNA preparations from positive field samples. The system was designed to amplify a 127 nucleotide-long RNA region located within the vp60 gene, based on the alignment of six sequences originated in Portugal, obtained in our laboratory, and 11 sequences from France and Italy. The primers and probe target sequences are highly conserved in the vast majority of the RHDV2 sequences presently known. In the sequences showing variability, only one mismatch is found per strain, usually outlying the 3′ end of the primer or probe hybridization sequences. The specificity of the method was demonstrated in vitro with a panel of common rabbit pathogens. Standardization was performed with RNA transcripts obtained from a recombinant plasmid harboring the target sequence. The method was able to detected nine RNA molecules with an efficiency of 99.4% and a R2 value of 1. Repeatability and reproducibility of the method were very high, with coefficients of variation lower than 2.40%. The assay was proven a valuable tool to diagnose most of RDVH2 circulating strains, and may be also useful to monitor viral loads, and consequently, disease progression and vaccination efficacy.
Type: article
Appears in Collections:MVT - Publicações - Artigos em Revistas Internacionais Com Arbitragem Científica
MED - Publicações - Artigos em Revistas Internacionais Com Arbitragem Científica

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