Computational protocol: Molecular identification of Giardia and Cryptosporidium from dogs and cats

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Protocol publication

[…] All PCR products from the molecular assays for both Giardia cysts and Cryptosporidium oocysts were cut out of agarose gels and purified with QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The purified PCR products were directly sequenced in both directions on an ABI Prism 3100 (Applied Biosystems, Japan) Genetic Analyser using a Big Dye Terminator V.3.1 cycle sequencing kit (Applied Biosystems, Japan). The accuracy of the data was confirmed by two directional sequencing of the obtained sequences and by the alignment of the nucleotide sequences of the GDH gene for Giardia cysts and the SSU rRNA for Cryptosporidium oocysts against reference sequences retrieved from GenBank using the program ClustalW. Sequence similarity was also determined using the Basic Local Alignment Search Tool (BLAST) and the phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5. The evolutionary history was inferred using the Neighbor-Joining method [] and the respective evolutionary analyses were conducted using MEGA 5 []. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches in []. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method [] and are in the units of the number of base substitutions per site.Figure 1. The nucleotide sequences of the positive samples have been submitted to the GenBank and they are available in the GenBank database under the Accession Nos. EU583462–EU583467 and EU591705 (93%–97% homology) for Giardia and EU606193 (94% homology) and EU606194 (99% homology) for Cryptosporidium. […]

Pipeline specifications

Software tools Clustal W, BLASTN, MEGA
Organisms Canis lupus familiaris, Felis catus, Homo sapiens, Cryptosporidium parvum
Diseases Infection, Pancreatic Cyst, Coinfection