A new diagnostic method for the detection of endogenous Rous-associated virus-type provirus in chickens

A quick and simple method has been developed to detect the presence or absence of the endogenous Rous-associated virus (RAV) element ev1 in chickens. The procedure consists of a one-tube multiplex polymerase chain reaction (PCR) involving three oligonucleotide primers that are specific for the upstream flanking region, the long terminal repeat (LTR), and the downstream flanking region of the proviral insert, respectively. The multiplex reaction allows for the unambiguous discrimination between ev1+/ev1+ homozygote, ev1-/ev1- homozygote, and ev1+/ev1- heterozygote birds. The method works best with purified genomic DNA as substrate, but can also be used with rapidly prepared, "crude" DNA samples. The combination of speed with the safety of a nonradioactive procedure, and the ability to perform large numbers of assays by a semi-automated procedure, make this method attractive for large-scale screening projects. The ev1 locus has been used as a model system to demonstrate the feasibility of the PCR diagnostic approach. However the same principle should be applicable to the analysis of other RAV-type ev loci, as well as endogenous elements belonging to other families of viruses as sequence information for the flanking regions of these inserts becomes available.