Development of a primer-probe energy transfer based real-time PCR for detection of Marek's disease virus.
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.
Barfoed
Annette Malene
AM
Danish Medicines Agency, Axel Heides Gade 1, DK-2300 Copenhagen S, Denmark. amba@dkma.dk
Østergaard
Erik
E
Frandsen
Peer Lyng
PL
Nielsen
Eva Baekdahl
EB
Sandberg
Eva
E
Rasmussen
Thomas Bruun
TB
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
D036022:Capsid Proteins; D015345:Oligonucleotide Probes