Abiprism 3130 genetic analyzer automated sequencer

PCR products were purified with the Wizard SV Clean-up System (Promega). The final DNA concentration was estimated by comparison with a DNA Ladder Marker (Promega) in 2 % agarosegel. All amplicons were directly sequenced by targeting cox1 gene in both directions using the mentioned primers by ABIPRISMTM 3130 Genetic Analyzer automated sequencer (Applied Biosystem, USA). Ambiguous (heterozygous) sites were coded using the standard IUPAC codes for combinations of two or more bases. Contigs from all samples were aligned, justified and edited in consensus positions compared to GenBank sequences of all regional species using Sequencher Tmv.4.1.4 Software for PC (Gene Codes Corporation). The diversity testes of analyzed sequences (Haplotype diversity; Hd and Nucleotide diversity: Pi) were determined by DnaSP 5.10.1 software [49 (link)].

To confirm the specificity of the LAMP primers, a single round PCR was done based on B3 and F3 outer primers. Amplicons of the ITS2 gene were purified with the Wizard SV Cleanup System (Promega). ABIPRISMTM 3130 Genetic Analyzer automated sequencer (Applied Biosystem, USA) directly sequenced PCR products from 10 randomly selected samples. Contigs (overlapped sequences) from all samples were aligned and edited at consensus positions compared to GenBank sequences of all regional species using Sequencher Tmv.4.1.4 Software for PC (Gene Codes Corporation). T. canis sequences of southwest Iran (Accession nos.; AB743614-AB743617 and AB819327-AB819330) were directly retrieved from GenBank database (FASTA format). The sequences pairwise distances (percent identity and divergence) between sequenced isolates and other country sequences were constructed using the MegAlign program from Laser Gene Bio computing Software Package (DNASTAR, Madison, WI). MEGA 5.05 software with maximum likelihood algorithm and Kimura2-parameter model were used in order to construct phylogeny tree. The diversity (haplotype and nucleotide diversity), neutrality indices (Tajima’s D and Fu’s Fs statistic) and fixation index (Fst) were estimated by DnaSP software version 5.10 [24 ].