Molecular Investigation for Lymphocystis Disease Virus (LCDV)
Subjects with nodules or skin alterations (n = 9) were analysed for the Lymphocystis disease virus (LCDV) by molecular method. The skin tissue or a portion of the sampled fin was subjected to DNA extraction using approximately 20 mg of tissue. DNA was extracted using the Purelink genomic DNA extraction kit (Invitrogen, USA) according to the manufacturer’s directions. The LCDV investigation was then conducted on these samples by a nested PCR assay. A first amplification step was performed using the LF7/LC1R primers (Kitamura et al., 2006; Kvitt et al., 2008), and a second amplification step using the LCDV qPCR F1 and LCDV qPCR R3 primers (Ciulli et al., 2015). For each amplification, a reaction mix containing 1X PCR Buffer, 1 mM MgCl2, 200µM dNTP, 0.4 µM of each primer, 1.25U of Taq polymerase and 5µl of DNA in a final volume of 25 µl was performed. The first amplification step was conducted with 94 ° C for 5 minutes, 45 cycles consisting of denaturation at 94 ° C for 2 minutes, annealing at 55 ° C for 2 minutes, extension at 72 ° C for 2 minutes and a final extension at 72 ° C for 10 minutes. The second amplification step was conducted at 95 ° C for 5 minutes, 45 cycles consisting of denaturation at 95 ° C for 15 seconds, annealing at 50 ° C for 30 seconds, extension at 72 ° C for 30 seconds and a final extension at 72 ° C for 10 minutes. The result of nested PCR was visualized by electrophoresis on 1.5% agarose gel by running the samples together with a reference molecular marker (100 bp ladder, Invitrogen, USA).