Computational protocol: Prevalence and Sequence-Based Identity of Rumen Fluke in Cattle and Deer in New Caledonia

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Protocol publication

[…] Genomic DNA was extracted from individual fluke specimens using the DNEasy Blood and Tissue Kit (QIAGEN, Germany), according to the manufacturer’s recommendations. The ITS-2 region of the rDNA gene, plus the flanking 5.8S and 28S sequences, were amplified by PCR using the generic trematode primers, ITS-2For 5’-TGTGTCGATGAAGAGCGCAG-3’ and ITS-2Rev 5’-TGGTTAGTTTCTTTTCCTCCGC-3’, as described by Itagaki et al. (2003) []. PCR was conducted in 25 μl reaction volumes comprising of 1μl genomic DNA template, 12.5 pmol of each primer (Eurofins, Germany), 0.2 mM of each dNTP (Invitrogen, USA), 2 mM MgCl2 and 2.5 U Platinum Taq polymerase in 1× Platinum Taq buffer (Invitrogen, USA). The PCR was carried out on an Applied Biosystems 2720 PCR machine under the following conditions: 95°C for 10 min; 35 cycles of 94°C for 1 min; 53°C for 1 min; 72°C for 1 min; followed by 72°C for 10 min. PCR products were separated on a 1.2% agarose gel prepared in Tris–acetate–EDTA (TAE) buffer incorporating GelRed (Cambridge Bioscience, UK) and visualised on a UV transilluminator. PCR products of the appropriate size (~500 bp) amplified from individual rumen fluke were purified using QIAquick PCR Purification Kit (QIAGEN, Germany) as specified by the manufacturer. PCR products were eluted using 30μl Elution Buffer and supplied to MWG Eurofins (Germany) at a concentration of 5ng/μl, in 15μl, along with the ITS-2For primer, for direct nucleotide sequencing. The chromatogram files obtained for the forward and reverse sequences were aligned using Lasergene 10 core suite Software SeqMan Pro (DNASTAR, USA) to assess the quality of the sequences and assemble the full-length ITS-2 fragment in order to compare with reference sequences in GenBank using BLASTn at the European Bioinformatics Institute website (http://www.ebi.ac.uk/).Paramphistomes which were identified as Calicophoron calicophorum were further analysed through amplification of the mitochondrial DNA encoding transfer RNA (tRNA-Thr/Cox1) region using the primers described by Martínez-Ibeas et al (2013) []: tRNA-ThrFor 5’-TGGAGAGTTTGGCGTCTTTTT-3’ and tRNA-ThrRev 5’-CCATCTTCCACCTCATCTGG-3’. The 25 μl reaction contained 2μl of DNA template, 20 pmol of each primer, 0.2 mM of each dNTP, 2.5 mM MgCl2 and 0.1U Platinum Taq polymerase in 1× Platinum Taq buffer. The thermocycler was programmed as follows: 92°C for 2 min; 38 cycles of 95°C for 60 secs; 65°C for 30 secs; 72°C for 90 secs; followed by 72°C for 10 min. The amplified product of 885 bp was visualised and sequenced as previously described. Two forward and one reverse sequence for each individual were obtained for alignment using Lasergene 10 core suite software SeqMan Pro (DNASTAR, USA) to provide a consensus. The tRNA-Thr/Cox1 sequences were aligned by MUSCLE using Alignment Explorer in MEGA6 [] and trimmed to equal lengths. A Neighbour-Joining phylogenetic tree was constructed in Tree Explorer to assess the level of heterogeneity. A published Calicophoron daubneyi sequence from the same locus was used as outgroup (Accession number: KJ574046.1, []). Tree reliability was assessed by the bootstrap method with 1000 pseudoreplicates. […]

Pipeline specifications

Software tools DNASTAR Molecular Biology Suite, BLASTN, MUSCLE, MEGA
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Bos taurus, Fischoederius elongatus, Orthocoelium streptocoelium