Development and initial validation of quantitative real-time PCR (qPCR) assays for the three serotypes of Marek's Disease Virus (MDV) are described. Also described is the development of an internal control qPCR assay that detects the chicken α2(VI) collagen gene. To reduce costs, a duplex assay for MDV1 and the internal control was also developed. The MDV qPCR assays were specific to their target gene and more sensitive than standard PCR when compared using Australian field and vaccine strains of MDV. All assays were found to have acceptable reproducibility. Relative abundance (RA) of MDVI and HVT viruses was quantified using the relative standard curve method in twenty experimentally infected chickens over a period of 7-35 days post infection and was shown to vary during the course of the infection. These qPCR assays will be useful for reliable differentiation and quantitation of MDV for a variety of applications.

Methods for Taqman quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and inter-assay coefficients of variation of less than 3% for Ct and less than 21.5% for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an attenuated strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample.

Methods for taqman real-time PCR assays to detect the three serotypes of MDV are available (Islam et al., 2004), and an absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Thus, it is now possible to perform qPCR assays for all three serotypes ofMDV on a sample. Absolute quantification of MDV2 in dust samples from poultry farms across Australia in a preliminary study, revealed the presence of MDV2 in 13 of 30 samples tested.

Quantitative real-time PCR (qPCR) assays for the three serotypes of Marek's disease virus (MDV) have been developed. An internal control qPCR assay that detects chicken α2 (VI) collagen gene was also developed to allow quantitation of MDV. To reduce costs and time, the assays for MDV1 and the internal control were combined into a duplex assay. The sensitivity, specificity, precision, and reproducibility of each assay are reported. The MDV qPCR assays were specific to their target gene when compared using Australian field and vaccine strains of MDV and 10–100-fold more sensitive than standard PCR. Using DNA from infected spleen tissue, the lower limit of detection of total DNA (viral and host combined) was 0.025 ng for the MDV1 and collagen assays, and 0.25 ng for the HVT and MDV2 assays. All assays were found to be highly reproducible for Ct values, but less so for calculated concentrations. MDV1 and HVT were quantitated in spleen tissue of twenty experimentally infected chickens 7–35 days after infection. The relative abundance of MDV1 exhibited a clear peak at day 14 post-infection, whereas HVT displayed an increasing trend over the 35 days post-infection. The duplex assay was optimized such that it was able to accurately quantitate MDV1 in samples of very high, medium, and very low relative abundance of MDV1. These qPCR assays will be useful for reliable differentiation and quantitation of MDV for a range of research and industry applications.

Methods for Taqman quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and inter-assay coefficients of variation of less than 3% for Ct and less than 21.5% for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an attenuated strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample.