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).