title: A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2).
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 R(2) 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.
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Rua General Morais Sarmento, 1500-310 Lisbon, Portugal. Electronic address: margarida.duarte@iniav.pt.
authors.forename: Margarida Dias
authors.initials: MD
authors.lastname: Duarte
authors.affiliation: Universidade de Évora, ICAAM/Escola de Ciências e Tecnologia, Apartado 92, 7002-554 Évora, Portugal.
authors.forename: Carina L
authors.initials: CL
authors.lastname: Carvalho
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Rua General Morais Sarmento, 1500-310 Lisbon, Portugal.
authors.forename: Silvia C
authors.initials: SC
authors.lastname: Barros
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Rua General Morais Sarmento, 1500-310 Lisbon, Portugal.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Henriques
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Rua General Morais Sarmento, 1500-310 Lisbon, Portugal.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Ramos
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Rua General Morais Sarmento, 1500-310 Lisbon, Portugal.
authors.forename: Teresa
authors.initials: T
authors.lastname: Fagulha
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Rua General Morais Sarmento, 1500-310 Lisbon, Portugal.
authors.forename: Tiago
authors.initials: T
authors.lastname: Luís
authors.affiliation: Universidade de Évora, ICAAM/Escola de Ciências e Tecnologia, Apartado 92, 7002-554 Évora, Portugal.
authors.forename: Elsa L
authors.initials: EL
authors.lastname: Duarte
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária (INIAV), Rua General Morais Sarmento, 1500-310 Lisbon, Portugal.
authors.forename: Miguel
authors.initials: M
authors.lastname: Fevereiro
mesh_terms: D000818:Animals; D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000821:virology*; D017929:Hemorrhagic Disease Virus, Rabbit / Q000145:classification / Q000235:genetics*; D011817:Rabbits; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
keywords: RHDV2; RHDVb; Rabbit hemorrhagic disease virus; Real time RT-qPCR; Taqman RT-PCR