title: Development of a primer-probe energy transfer based real-time PCR for detection of Marek's disease virus.
abstract: A real-time PCR assay, which enables simultaneous detection and differentiation of all three serotypes of Marek's disease virus, without the need for post-PCR sequencing, has been developed. The assay is based on the primer-probe energy transfer real-time PCR, which has a relatively high tolerance towards point mutations in the probe region. The PCR is followed by a probe melting point analysis, which enables confirmation of identity of amplicon and differentiation of serotypes. The assay targets the MDV031 gene, encoding UL19 major capsid protein-like protein and was shown to be quantitative, with a detection limit below 10TCID(50)/ml starting material. This sensitivity is similar to the one obtained with traditional virus cultivation. However, the PCR method can provide a laboratory result within a day, while the virus cultivation method takes more than a week to perform. The new method will be useful for testing of avian live viral vaccines and screening for extraneous agents.
authors.affiliation: Danish Medicines Agency, Axel Heides Gade 1, DK-2300 Copenhagen S, Denmark. amba@dkma.dk
authors.forename: Annette Malene
authors.initials: AM
authors.lastname: Barfoed
authors.forename: Erik
authors.initials: E
authors.lastname: Østergaard
authors.forename: Peer Lyng
authors.initials: PL
authors.lastname: Frandsen
authors.forename: Eva Baekdahl
authors.initials: EB
authors.lastname: Nielsen
authors.forename: Eva
authors.initials: E
authors.lastname: Sandberg
authors.forename: Thomas Bruun
authors.initials: TB
authors.lastname: Rasmussen
mesh_terms: D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics; D004735:Energy Transfer; D025922:Mardivirus / Q000235:genetics / Q000302:isolation & purification*; D008380:Marek Disease / Q000175:diagnosis*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D044366:Transition Temperature
chemical_list: D036022:Capsid Proteins; D015345:Oligonucleotide Probes