DNA hybridization procedure to detect pseudorabies virus DNA in swine tissue

A DNA hybridization technique was developed to detect the presence of pseudorabies virus (PRV) DNA. P Nick translated probes of high specific activity were prepared from transformed Escherichia coli plasmids into which Bacillus amyloliquefaciens H (Bam H1) restriction fragments of PRV DNA had been inserted. Swine cellular DNA and tissue culture PRV DNA were digested with Bam H1, separated by agarosegel electrophoresis, transferred onto nitrocellulose paper, hybridized to the radioactive probes, and washed under high stringency conditions; autoradiographs were then prepared. Under the optimal hybridization conditions described, the detection limit of these probes was 10(-11)g of PRV DNA. In reconstruction experiments, 3 of the selected probes cross hybridized with digested swine cellular DNA, and 4 probes did not. The addition of polyuridylic acid and polyguanylic acid to the hybridization reactions did not alter the amount of hybridization. The results indicated that this procedure may be useful for studying the latency of pseudorabies viral infection.