Development of a quantitative-competitive polymerase chain reaction assay for serotype 1 Marek's disease virus

We have developed a quantitative-competitive (QC) polymerase chain reaction (PCR) for the detection of Marek's disease virus (MDV) DNA. The assay util izes a competitor DNA that differs from the viral DNA of interest by having a small insertion. The competitor DNA acts as an internal standard for the estimation of viral DNA in an unknown sample. The amount of viral DNA in a sample is quantitated by coamplification in the presence of a known amount of competitor DNA. The same PCR primers that amplify the viral DNA also am plify the competitor DNA. When the amount of competitor is equal to the: am ount of viral DNA in a sample, there is equal amplification of the competit or and the virus. Thus, we are able to quantitate the viral DNA in an unkno wn sample. To establish the utility of this assay, in vivo correlations bet ween virulence and virus replication were studied. Our data demonstrated th at a more virulent strain of MDV (648A) replicated better in thymus during cytolytic infection than did a less virulent strain (GA). However, no diffe rences in virus titer were observed when these two viruses were propagated in tissue culture. Our data are consistent with the generally held idea tha t "hot" strains of MDV replicate earlier and better in birds. Thus, QC-PCR is extremely specific and sensitive to measure MDV DNA over a wide range an d can be applied to in vivo studies of viral pathogenesis.