ESTABLISHMENT OF CONDITIONS FOR THE DETECTION OF BOVINE HERPESVIRUS-1BY POLYMERASE CHAIN-REACTION USING PRIMERS IN THE THYMIDINE KINASE REGION

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antise nse primers in the thymidine kinase (TK) coding region. The amplificat ion product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). T he sensitivity of four methods of sample preparation which are standar d DNA extraction, modified proteinase K (PK) digestion, GeneReleaser(T M) + 34 cycles or + 44 cycles, and boiling were compared with virus is olation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturatio n of samples and cooling in ice prior to PCR were essential for the am plification of BHV-1 DNA for samples prepared by standard DNA extracti on and modified PK digestion. The preparation of samples by GeneReleas er(TM), a proprietary nucleic acid releasing cocktail, showed 10 to 1, 000-fold increase in sensitivity compared to standard DNA extraction a nd modified PK digestion. No amplification was observed in samples pre pared by boiling. The sample preparation of BHV-1 LA strain by GeneRel easer(TM) showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaser(TM) has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells usin g either ethidium bromide stained gel or Southern blot hybridization.