Computational protocol: RT-PCR Assays for Seven Serotypes of Epizootic Haemorrhagic Disease Virus & Their Use to Type Strains from the Mediterranean Region and North America

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Protocol publication

[…] For confirmation of RT-PCR typing results, amplified cDNA products from Seg-2 were gel-purified and sequenced directly on an Applied Biosystems BigDye ddNTP 3730 capillary sequencer, using the PCR primers, as sequencing primers. The resultant sequences were analysed and aligned using the Clustal W program and MAFFT (www.ebi.ac.uk/Tools/mafft/) and then subjected to either BLAST analysis and/or in silico comparisons to Seg-2 sequence data available for the 7 EHDV serotypes (including both western and eastern strains). Phylogenetic comparisons were made using Neighbour-Joining (NJ) algorithms and the trees were constructed in MEGA ver. 4.1 software , using nucleotide sequences in a pair-wise deletion, p-distance algorithm, and bootstrapped using 500 replicates. […]

Pipeline specifications

Software tools Clustal W, MAFFT, MEGA
Application Phylogenetics
Organisms Bos taurus, Homo sapiens
Diseases Bluetongue, Virus Diseases
Chemicals Etoposide, Oligonucleotides