Computational protocol: Experimental infection of Pacific oyster Crassostrea gigas spat by ostreid herpesvirus 1: demonstration of oyster spat susceptibility

Similar protocols

Protocol publication

[…] OsHV-1 isolates were characterized by sequencing PCR products using primer sets targetting two regions of the virus genome, the region IA (ORF43) that encodes a putative apoptosis inhibitor and the region C (ORF4) that encodes two proteins of unknown function []. The first primer pair used IA1/IA2 (IA1: CGC GGT TCA TAT CCA AAG TT/IA2: AAT CCC CAT GTT TCT TG CTG) amplified a 607 bp fragment and the second primer pair, C2/C6 a (C2: CTC TTT ACC ATG AAG ATA CCC ACC/C6: GTG CAC GGC TTA CCA TTT TT) amplified a 709 bp fragment. The sequencing reaction was carried out into a 10 μL final volume, containing 1.8 μL of sequencing buffer, 0.4 μL of BigDye® Terminator v3.1 (Applied Biosystems; Carlsbad, USA), 1.5 μL of primer Forward or Reverse at 4 μM, fresh purified PCR products and 10 μL sterile DNAse and RNAse free water. The program consisted of an initial denaturation of 3 min at 96°C followed by 35 cycles of 30 s at 96°C, 30 s at 50°C and 4 min at 60°C. This reaction was performed using a 96-well plate. Sequencing reactions were then purified as follows. In each sample, 60 μL of 100% ethanol were added and samples were centrifuged at 1500 g for 30 min. The plate was inverted to remove ethanol. Then, 60 μL 70% ethanol were added, followed by centrifugation of the plate at 1000 g for 10 min. The plate was then centrifuged upside down for 30 s to remove ethanol. Finally, samples were dried in a Speed Vac and re-suspended with 10 μL deionized formamide. Samples were loaded in ABI PRISM® 3130 XL-Avant Genetic Analyzer (Applied Biosystems), using a 36 cm capillary array and POP 7 polymer. Sequences were edited with Chromas lite 2.01 version software. A multiple alignment was achieved by Bioedit 7.0.9 version software using the algorithm CLUSTAL W. Sequence results were compared to the consensus sequence OsHV-1 available on GENBANK [accession number AY509253]. […]

Pipeline specifications

Software tools Chromas, BioEdit, Clustal W
Application Sanger sequencing
Organisms Ostreid herpesvirus 1, Crassostrea gigas
Diseases Infection