Computational protocol: Endoparasites in the feces of arctic foxes in a terrestrial ecosystem in Canada

Similar protocols

Protocol publication

[…] Genomic DNA was extracted from 300 μL of fecal suspension () using the Fast-DNA kit (MP Biomedicals) with Lysis Matrix E beads (MP Biomedicals), followed by the PCR Purification kit (Qiagen). Coccidian species in each fecal sample were detected using a universal coccidia primer cocktail designed to amplify a ∼315-bp region of 18S rDNA in a real-time quantitative PCR assay and differentiated using melt-curve analysis (). The PCR product melting temperature (Tm) is based on the nucleotide sequence composition, length, and G–C content, so genetically distinct members of the same genus or species can be differentiated by the melt curve shape and Tm and identified by comparison to in-run controls and/or sequencing (). All qPCR analyses were performed using the CFX96 Real-Time PCR detection system (Bio-Rad Laboratories) as described previously () except 1X Evagreen Supermix (Bio-Rad Laboratories) was used in the final reaction mix. Each PCR reaction plate included two negative control wells (water), a standard curve consisting of plasmid DNA from Eimeria bovis (105–101 oocysts), and wells containing DNA from Toxoplasma gondii (genomic, ATCC, Virginia, USA), Cystoisospora sp. (plasmid, in house), Neospora caninum (genomic, ATCC, Virginia, USA), Sarcocystis cruzi (genomic, in house), and E. bovis (plasmid, in house) as positive controls.Melt curves from positive samples were visually compared to the controls for preliminary identification, and amplified DNA from positive reactions were sequenced by Macrogen (Seoul, South Korea) for confirmation using original primers. Forward and reverse sequences were assembled with PreGap4 and Gap4 (Staden Package) and consensus sequences were compared with reference sequences in GenBank™ using the nucleotide Basic Local Alignment Search Tool (BLASTN). Multiple alignments of reference and sample sequences were performed with CLUSTAL X. Neighbor-joining phylogenetic trees and branch reliability bootstrap values (100 iterations) were constructed with the program PHYLIP 3.69 (). […]

Pipeline specifications

Software tools BLASTN, Clustal W, PHYLIP
Application Phylogenetics
Organisms Vulpes lagopus, Hemisus marmoratus, Toxascaris leonina
Diseases Gastrointestinal Diseases