2.6 PCR and Sequencing of RPO30 gene for phylogenetic analysis:
PCR amplification of RPO30 gene was carried out by using the specific primers (Table 1 ) and sequenced using Sanger’s sequencing method. The sequence was submitted to NCBI GenBank and obtained accession number MW590715. The reference sequences of LSDV from China, Bangladesh, Kenya, Indian states of Odisha and Haryana, GTPV and SPPV virus strains retrieved from GenBank and the phylogenetic analysis of RPO30 gene generated in present investigation. The sequences were aligned and compared using BioEdit and the phylogenetic analysis was carried out using MEGA-X.
TaqManTM Probe Real-Time PCR Assay:
For this assay, 25µl reaction mixture containing 12.5µl of 2X TaqManTM Fast Universal PCR Master Mix, 1µl of each forward (f2) and reverse (r33) primers, 1µl of TaqManTM probe was prepared. 150ng of template DNA was added to the reaction mixture and nuclease-free water was used to make up the total volume of 25 µl. The polymerase chain reaction was carried out in Real-time PCR system (Applied Biosystems). The conditions for real-time PCR were as follows; 50oC for 2 min for Uracyl -D- glycosylase (UDG) activation, 1 cycle at 95oC, for 10 min for DNA polymerase activation, 40 cycles of 95oC for 15s of denaturation and 60oC for 1 min of annealing and extension. One positive control DNA from goat pox vaccine and one negative control (NTC) were also included in the reaction panel.
Results