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.