Molecular detection using conventional Polymerase Chain Reaction
Fragments containing the B2L genes were amplified by PCR in a 25 μl reaction volume containing 20UM forward primer, 20 UM reverse primer, 2 mM dNTPs, 5x PCR Buffer (Applied Biosystem), 5X Taq Polymerase (Jena Biosciences) and 5 μl template DNA. Cycling conditions were: initial denaturation at 94°C for 3 mins, followed by 35 cycles at 94°C for 50s, 52°C for 60s and 72°C for 90s, and final extension at 72°C for 7 min. Primers ORFV-B2Lf-For 5’-GACCTTCCGCGCTTTAATTT-3 and ORFV-B2Lf-Rev 5’-CCCGCCTGCTAAAAGACT-3’. Each PCR run included a negative control consisting of 5μl of PCR grade water instead of the DNA template. Amplified PCR products were subjected to electrophoresis on 1.5% agarose gel stained with ethidium bromide and visualized using Biorad® transilluminator.