Polymerase chain reaction (PCR) was evaluated for the early detection of Aujeszky´s disease virus (ADV) DNA from virus-infected cell cultures. For the purpose, the Korean ADV NYJ1-87 was propagated in swine kidney (SK) cells and subjected to the amplification of DAN (217 bp) by PCR using sense and antisense primers specific to gp50 gene of the ADV.
In detection of cell-associated viral DNA, reliable PCR conditions were determined as 30 cycles of reaction consisting 1 minute results with reagent mixtures of 50 ㎕ containing 200 uM dNTPs, 0.2 uM each sense and antisense primers, 1 mM MgCl₂and 10% (v/v) template DNA in the final concentrations. ADV-specific DNAs were detected as early as 6, 6, and 9 hours post-infection, respectively, from lysates of the SK cells infected with ADV of 10³, 10² and 10¹ TCID_50/ml by this condition. In culture supermatant, the DNAs were detected from ADV of as low infectivity as 10^-3 TCID_50/mlby the reduced reagent concentrations and 30 cycles of 1 minute each of denaturation at 94˚C and annealing at 55˚C, and 2 minutes of polymerization at 72˚C. The lowest amount of detectable ADV DNA was 1 fg. In conclusion, the PCR condition established in the present study was recognized as a feasible alternative to time-consuming procedures in isolation and characterization of the virus.
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