Computational protocol: Effect of oral administration of Kudoa septempunctata genotype ST3 in adult BALB/c mice

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Protocol publication

[…] Quantitative real-time PCR was used to detect K. septempunctata 18S ribosomal DNA []. DNA was extracted from various organs and the stool suspension filtrates using a QIAamp DNA Mini Kit (Qiagen, Venlo, Netherlands), following the manufacturer’s protocol. qPCR was performed using TaqMan Universal Master Mix (Applied Biosystems, Carlsbad, CA, USA). The primers and probes described by Kawai et al. [] were used. All reactions were carried out using the StepOne™ system (Applied Biosystems) with an initial denaturation step (10 min, 95 °C) and 50 cycles of amplification consisting of 95 °C for 15 s and 60 °C for 60 s. The gene copy number was determined using the standard curve method with plasmid DNA containing a copy of the target gene as a control.Conventional PCR primers were designed to detect two K. septempunctata mitochondrial genes: cytochrome c oxidase subunit I (cox1) and large subunit rRNA (rnl) []. Amplification was performed on a Mycycler thermocycler (Bio-Rad, Hercules, CA, USA) with Diastar™ Taq DNA polymerase (SolGent Co. Ltd., Daejeon, Korea) using the protocol of Takeuchi et al. []. Negative controls (without template DNA) were included to check for contamination. The PCR products were sequenced on an ABI 3730XL DNA analyser. Mitochondrial genes were subjected to a multiple sequence alignment using ClustalW (http://www.clustal.org) with MEGA v. 5.1. New sequences were deposited in GenBank as KU163620 and KU163621. […]

Pipeline specifications

Software tools Clustal W, MEGA-V
Application GWAS
Organisms Mus musculus, Kudoa septempunctata, Paralichthys olivaceus