Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR.
A real-time PCR method was developed, optimised and validated, to enable quantitation of Marek's disease virus genomes as copy number per million host cells. The duplex PCR measured the virus meq gene and host ovotransferrin gene in a single reaction enabling correction for differences in amount of sample DNA added. A bacterial artificial chromosome (BAC) clone of the virus genome, and a plasmid (pGEM-T-ovo) bearing a fragment of the chicken ovotransferrin gene, were used to quantify virus and host genomes respectively. This sensitive and reproducible assay was established initially using chicken lymphocyte DNA, then adapted for feather tip DNA by inclusion of bovine serum albumin in the reaction to overcome inhibition by melanin. The principal advantages are: (1) determination of absolute virus genome copy number enabling meaningful comparison between samples; (2) expression of copy number per million cells, allowing direct correlation with plaque assays; (3) using BAC-cloned whole virus genome as a standard potentially enables any virus gene to be used as the PCR target. This is the first report of quantitation of MDV genomes in feather tips, and application of this assay could significantly further our understanding of pathogenesis, spread, diagnosis, genetic resistance and vaccinal control of Marek's disease.
Baigent
Susan J
SJ
Viral Oncogenesis Group, Institute for Animal Health, Compton, Berkshire RG20 7NN, UK. sue.baigent@bbsrc.ac.uk
Petherbridge
Lawrence J
LJ
Howes
Ken
K
Smith
Lorraine P
LP
Currie
Richard J W
RJ
Nair
Venugopal K
VK
D000818:Animals; D002460:Cell Line; D002645:Chickens / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis*; D005241:Feathers / Q000821:virology*; D005260:Female; D018628:Gene Dosage; D016679:Genome, Viral; D008214:Lymphocytes / Q000821:virology*; D008297:Male; D025922:Mardivirus / Q000235:genetics*; D008380:Marek Disease / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods
D004279:DNA, Viral