2.5 PCR amplification of MDV oncogenes and sequencing
Out of 120 MD suspected tissue samples investigated from 13 flocks, 17 samples from 5 flocks were found to be MD positive by Meq gene specific PCR. MD positive samples were used for the amplification of MDV oncogenes, Meq, vIL-8 and pp38. The primer sequences used are provided in supplementary Table 2. The PCR amplification of Meq and vIL-8 genes were carried out as previously described with slight modifications (Tian et al., 2011). The PCR amplification was carried out using 4 µl of template DNA in a total volume of 50 µl reaction volume containing 5 µl of 10x Taq buffer with MgCl2, 2 µl of each primer (20 pmol), 1 µl (10 mMol) of dNTPs and 0.5 µl (5 U/ml) of Taq DNA polymerase and nuclease free water. The PCR conditions for Meq and vIL-8 were as follows: 94˚C for 4 min, 35 cycles of 94˚C for 1 min, 56˚C for 1 min, 72˚C for 1 min and final elongation at 72˚C for 10 min. PCR amplification of pp38 gene was carried out as per the method of Zhuang et al. (2015). The amplified products were checked in 1.5% agarose gel and PCR product was purified. The products were sequenced by Sanger sequencing. The obtained sequences were edited and aligned in EditSeq and MegAlign program of DNAStar software. The sequences of the field MDV strains were submitted to NCBI database (Table 1).