Qiaexii pcr purification kit

Samples found positive by qRT‐PCR were selected for PCR and sequencing. The viral RNA was reverse transcribed and the C‐terminus of the N‐gene was amplified as previously described (Baazizi et al., 2017). The PCR amplicons were purified using the QIAEXII PCR purification kit (Qiagen) according to the manufacturer's instructions and sent to a commercial company (REFGEN, Ankara, Turkey) for sequencing. Sequences were assembled and analysed using SeqMan II (DNAStar Lasergene 8.0). Alignments of the N‐gene sequences were made using the Clustal W program and used for construction of distance matrices using the Kimura 2‐parameter nucleotide substitution model (Kimura, 1980) as implemented in the programme MEGA 6.0 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). A maximum‐likelihood phylogenetic tree was then generated using MEGA 6.0, and the robustness of tree topology was assessed using 1,000 bootstrap replicates.

The viral RNA was reverse transcribed using superscript III first strand synthesis kit (Invitrogen). The C-terminus of the N-gene was amplified using primer pairs NP3/NP4 [31 (link)]. Similarly the partial F-gene was amplified using primer pairs F1/F2 [32 (link)]. The PCR amplicons were purified using the QIAEXII PCR purification kit (Qiagen) according to the manufacturer’s instructions and sequenced using BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA) using the PCR primers. Sequences (from the ABI 3730 machine) were assembled and analysed using SeqMan II (DNAStar Lasergene 8.0). Nucleotide sequences of the viruses were aligned using the CLUSTAL X multiple sequence alignment program [33 ].
Full length genomic RNA sequencing was performed using a hemi-nested RT-PCR as described previously [34 (link)]. Initial first-strand synthesis and first round PCR was performed as above except first-strand synthesis was performed at 48°C and an annealing temperature of 55°C was used. Second round amplification was accomplished using KOD hot-start polymerase kit (Novagen). Sequencing of the terminal 5`and 3`ends of the PPRV genome was accomplished via RACE, as previously described [34 (link)].