Multiplex PCR and Sanger Sequencing for cox1 Gene Identification

To verify the multiplex PCR resulting from genotyping, 59 samples were selected to sequence the gene target of cox 1 randomly from the cysts obtained from all various species of animals. The PCR reaction was conducted using a thermal cycler (ABI, USA) in a 20 μl final volume containing 50–100 ng gDNA, 0.2 mM dNTPs (Ampliqon, Denmark), 1.5 mM MgCl₂ (Ampliqon, Denmark), 10 pmol of each primer (ordered from Pishgam Company), 1.5 U Taq DNA polymerase, and ddH₂O up to the final volume. Amplification was done using the specific primer pair for cox 1 by JB3: 5′-TTTTTTGGGCATCCTGAGGTTTAT-3′ and JB4.5: 5′-TAAAGAAAGAACATAATGAAAATG-3′ [84 (link)] resulted in an amplicon fragment of 450 bp in length. The temperature conditions were an initial denaturation at 94 °C for 5 min; then 35 cycles of denaturation at 94 °C for 45 s, annealing at 57 °C for 45 s, and elongation at 72 °C for 45 s. The final extension was done at 72 °C for 5 min. PCR products were detected in 1% agarose gel electrophoresis (Akhtarian, Tehran, Iran). The PCR product was excised from agarose gel, and sent to the Company (Pishgam Company, Tehran, Iran) for purification and sequencing. The sequencing results were analyzed using BLAST.