title: Development and validation of a real time PCR for the detection of myxoma virus based on the diploid gene M000.5L/R.
abstract: The myxoma virus (MYXV) causes severe infections in European rabbits that may reach mortality rates up to 100% depending on the viral strain. The typical symptoms and lesions induced by the virus are usually enough to permit the correct clinical diagnosis. However, in peracute forms the infection may be accompanied by unspecific symptoms. Sudden death may also occur without evident clinical signs of myxomatosis. Likewise, a clinical diagnosis of atypical forms of myxomatosis (amyxomatous) is often complicated and delayed due to the scarceness of skin lesions. As the disease control often depends on an early and unequivocal diagnosis of MYXV, laboratorial methods play a relevant role in the confirmation of MYXV infection. This study describes the development and validation of a novel, high accurate real time polymerase chain reaction assay (rtPCR) for the detection of MYXV. Primers were designed to amplify a 125-bp within the gene M000.5L/R, which is duplicated in the termini of the genome and is unique among Leporipoxvirus. The assay was negative for SFV and other poxviruses and was able to detect 2.6 copies of MYXV DNA proving the effectiveness, specificity and sensitivity of this diagnosis tool. The rtPCR has been applied successfully in INIAV laboratory for routine diagnosis of myxomatosis since 2005.
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, Laboratory of Virology, Rua General Morais Sarmento, 1459-011 Lisboa, Portugal. Electronic address: margarida.duarte@niav.pt.
authors.forename: Margarida Dias
authors.initials: MD
authors.lastname: Duarte
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, Laboratory of Virology, Rua General Morais Sarmento, 1459-011 Lisboa, Portugal. Electronic address: silvia.santosbarros@iniav.pt.
authors.forename: Sílvia Carla
authors.initials: SC
authors.lastname: Barros
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, Laboratory of Virology, Rua General Morais Sarmento, 1459-011 Lisboa, Portugal. Electronic address: margarida.henriques@iniav.pt.
authors.forename: Ana Margarida
authors.initials: AM
authors.lastname: Henriques
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, Laboratory of Virology, Rua General Morais Sarmento, 1459-011 Lisboa, Portugal. Electronic address: teresa.fagulha@iniav.pt.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Fagulha
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, Laboratory of Virology, Rua General Morais Sarmento, 1459-011 Lisboa, Portugal. Electronic address: fernanda.ramos@iniav.pt.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Ramos
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, Laboratory of Virology, Rua General Morais Sarmento, 1459-011 Lisboa, Portugal. Electronic address: tiago.luis@iniav.pt.
authors.forename: Tiago
authors.initials: T
authors.lastname: Luís
authors.affiliation: Instituto Nacional de Investigação Agrária e Veterinária, Laboratory of Virology, Rua General Morais Sarmento, 1459-011 Lisboa, Portugal. Electronic address: miguel.fevereiro@iniav.pt.
authors.forename: Miguel
authors.initials: M
authors.lastname: Fevereiro
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D005814:Genes, Viral; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D009233:Myxoma virus / Q000302:isolation & purification*; D009234:Myxomatosis, Infectious / Q000175:diagnosis*; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral
keywords: Leporids; Leporipoxvirus; Myxoma virus; Myxomas; Myxomatosis; Real time PCR