A FLUORESCENCE-BASED QUANTITATIVE PCR METHOD FOR INVESTIGATION OF PSEUDORABIES VIRUS LATENCY

A quantitative PCR method was developed in order to quantitate the num ber of copies of Pseudorabies virus (PRV) genome present in tissues fr om infected pigs. The method is based on the use of an internal standa rd that differs from the target DNA by a deletion of ten base pairs, a nd that is co-amplified with the target DNA. The resulting PCR product s are labelled with a fluorescent primer and are then separated and de tected by means of an automated sequencer. The assay was found to be s pecific and sensitive, allowing the detection of five copies of viral DNA among 10(6) host cells. The method was used successfully to quanti tate the number of PRV DNA copies in trigeminal ganglia samples from i nfected pigs during the acute and the latent stages of the infection. Between 12 and 3.10(5) copies of viral genome per 10(6) neuronal cells were detected in these tissues which is consistent with data publishe d previously.