Polymerase Chain Reaction for the Detection of Aujeszky's Disease Virus

Hwang, Dong-hee; Yeo, Sang-geon

Korean Journal of Veterinary Research 2003;43(2):239-246.

¹Plasma Fractionation Center, Korean Red Cross
²College of Veterinary Medicine.Institute of Animal Medicine Gyeongsang National University

오제스키병 바이러스 검출을 위한 Polymerase Chain Reaction

황동희¹, 여상건²

¹대한적십자사 혈장분획센터
²경상대학교 수의과대학.동물의학연구소

Abstract

Polymerase chain reaction (PCR) was evaluated for the early detection of Aujeszky's disease virus (ADV) DNA from virus-infected cell cultures. For the purposes, the Korean ADV NYJ1-87 was propagated in swine kidney (SK) cells and subjected to the amplification of DNA (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 each of denaturation at $94^{circ}C$, annealing at $55^{circ}C$ and polymerization at $72^{circ}C$. The PCR encountered best results with reagent mixtures of $50{mu}l$ containing $200{mu}M$ dNTPs, $0.2{mu}M$ each sense and antisense primers, 1 mM $MgCl_2$ and 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 $10^3$, $10^2$ and $10^1;TCID_{50}/ml$ by this condition. In culture supernatant, the DNAs were detected from ADV of as low infectivity as $10^ {-3};TCID_{50}/ml$ by the reduced reagent concentrations and 30 cycles of 1 minute each of denaturation at $94^{circ}C$ and annealing at $55^{circ}C$, and 2 minutes of polymerization at $72^{circ}C$. 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.

Key Words: ADV, PCR, gp50