Computational protocol: Giardia duodenalis in Alpine (Rupicapra rupicapra rupicapra) and Apennine (Rupicapra pyrenaica ornata) chamois

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Protocol publication

[…] A nested PCR was performed to amplify a 130 bp region from the SSU-rDNA gene, using the primers RH4 and RH11 for the first step, and the primers GiarR and GiarF in the second amplification round, as used for qPCR []. An additional analysis was carried out by using a semi-nested PCR to amplify a 432 bp fragment with the primers GDHeF and GDHiR in the primary reaction, and GDHiF and GDHiR in the secondary []. In all PCR reactions, positive (Giardia DNA) and negative (no template added) controls were added. All PCRs were carried out in a 25 μL volume containing 12.5 μL PCR master mix 2X (Promega), 5 μL template DNA, 0.6 mM of each primer and 0.1 mM BSA, 4 % dimethyl sulfoxide (DMSO), and were performed in a TProfessional Basic Thermocycler (Biometra GmbH, Göttingen, Germany). PCR products were visualized by electrophoresis on 1 % agarose gel stained by SYBR Safe DNA gel stain (Invitrogen). Amplicons were purified using the mi-PCR Purification Kit (Metabion GmbH). Both strands were sequenced by Bio-Fab Research s.r.l. (Rome, Italy). Sequences were edited with FinchTV 1.4 software (Geospiza, Inc, Seattle, WA, USA). To assign Giardia isolates to the correct assemblage, a comparison of the SSU-rDNA sequences by multiple alignments was performed using ClustalW2 software against known sequences available in GenBank. To test the significance of the results and to identify the sub-assemblage, a phylogenetic analysis was performed using MEGA6 software to compare the SSU-rDNA and gdh sequences with those of reference strains from different hosts. The best-fit model and parameters for tree construction were selected using the jModeltest software by the Akaike Information Criterion (AIC). […]

Pipeline specifications

Software tools FinchTV, Clustal W, MEGA, jModelTest
Applications Phylogenetics, Sanger sequencing
Organisms Giardia intestinalis, Rupicapra rupicapra, Rupicapra pyrenaica