Phylogeny of bovine norovirus in Egypt based on VP2 gene

Mohamed, Fakry F.; Ktob, Gamelat K.F.; Ismaeil, Mohamed E.A.; Ali, Ahmed A.H.; Goyal, Sagar M.

Initially, the one-step RT-PCR (QIAGEN, Valencia, CA, USA) was carried out to detect the BNoV RNA in tested fecal samples by targeting a conserved area in BNoV-RdRp gene (4543-5074) using the primer pair CBECu-F/R [18] . The PCR cyclic conditions were 50°C/30 min for reverse transcription, 95°C/15 min for PCR activation, and 35 cycles of 94°C/1 min for denaturation, 51°C/45 s for annealing, and 72°C/1 min for elongation followed by a final cycle of extension at 72°C/10 min. Then, another primer pair (BNoV-VP2-F/R) was designed to flank the BNoV-VP2 gene from base 6432 to base 7257 (826 bp) in the positive samples. The design of these primers was made based on the VP2 region of the GenBank reference sequence (Newbury2/UK/AF097917). The previous PCR cycle was used except for annealing that was changed to 55°C for 1 min. The PCR reactions were 25 μL in volume (1 μL enzyme mix, 12.5 μL buffer, 1 μL forward primer, 1 μL reverse primer, 2.5 μL of extracted RNA, and 7 μL RNase-free water). In negative controls, a 9.5 μL of RNase-free water was added with no RNA. The PCR amplicons were allowed to run through agarose gel by electrophoresis and then were visualized on UV transilluminator to visualize the band size. The oligonucleotide primers used in this study are listed in Table 1 .