Computational protocol: Genomic variability in Potato virus M and the development of RT-PCR and RFLP procedures for the detection of this virus in seed potatoes

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Protocol publication

[…] The CP gene amplicons generated in RT-PCR from test samples using primer PVM1/2 and PVM3/4 were purified using QIAquick PCR purification kit (Qiagne Inc., Mississauga, ON, Canada) following the procedures recommended by the provider and the dsDNAs amplified in a typical RT-PCR reaction (50 μl) were eluted with 30 μl of water (DNase-free and RNase-free). Purified PCR amplicons from the CP gene of PVM isolates CL1, CL3, CL4, Ca5, Ca128, Ca508 and Ca513 were sequenced in both orientations by automated cycle sequencing (York University, Toronto, ON, Canada) using primers PVM1/PVM2 and/or PVM3/PVM4 depending on the templates. Sequences of Canadian PVM isolates were compared with PVM sequences in the NCBI database with the program BLAST. Nucleotide and amino acid sequences were aligned using Clustal G (V1.1) and GeneDoc Multiple Sequence Alignment Editor and Shading Utility (V2.5.000)[]. The phylogenetic relationship of the Canadian potato isolates of PVM and 8 other known PVM strains/isolates (Table ) were deduced using the Bootstrap Neighbour-Joining (N-J) methods (random number generator seed: 111, number of bootstrap trails: 1000) in the Phylip formatted Clustal W (V1.82). The trees were visualized and the dendrograms were displayed using the program TreeView (V1.5). […]

Pipeline specifications

Software tools PHYLIP, Clustal W, TreeViewX
Application Phylogenetics
Organisms Solanum tuberosum, Potato virus M