2.5. Real-Time PCR

To assess the presence or absence of an amplicon with a simple yes/no answer, making it similar to a terminal PCR and gel electrophoresis, we tested primer sensibility and specificity in a protocol for Real-Time PCR (RT-PCR) technique. For some labs, it could be useful to assemble a reaction, load it into a single instrument, and obtain the needed information by visualizing species-specific amplification without the additional electrophoresis step. The DNA samples were used as a template for RT-PCR experiments performed in a Viia7 real-time PCR system (Applied Biosystems) at 1:10 dilution, with the primers described before [48]. The PCR volume of each sample was 10 μL with 5 μL of 2× RT-PCR SYBR Green Master Mix (Thermo Fisher), 0.05 or 0.1 pmol/μL for each primer and 2 μL of 1:10 diluted DNA template. The expected fragment of 291 bp in length had a theoretical melting temperature of about 87 °C calculated by Endmemo (http://www.endmemo.com/bio/tm.php). A slight difference in RT-PCR experiments is expected, due to the specific instrument used and the reaction conditions. In the set-up experiment, a specific amplicon with a melting temperature of about 80 °C was detected (data not shown), so we decided to test all fish specimens. Experiments were performed in triplicate. The diagram was elaborated with Excel.