Computational protocol: Detection of Toxoplasma gondii by PCR and Mouse Bioassay in Rodents of Ahvaz District, Southwestern Iran

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Protocol publication

[…] DNA was extracted from digested-tissue samples of brain, skeletal muscle, and liver using Bioneer Co., Seoul, South Korea, according to the manufacturer's instructions. Purified DNA concentration was determined by UV absorption [].For positive control, confirmed genomic DNA (Genbank accession number AB733004) was used in PCR amplification.Detection of T. gondii DNA was carried out using a single PCR assay targeting the GRA6 region. For T. gondii-specific amplification, two forward primer, 5′- GTAGCGTGCTTGTTGGCGAC-3′, and reverse primer, 5′-ACAAGACATAGAGTGCCCC-3′, primers were used as described by Fazaeli et al. [].The PCR reaction was performed in a 25 μL reaction mixture containing 10 pmol of each primer and 10 μL of extracted DNA, 75 mM Tris-HCl (PH 8.5), 20 mM (NH4)2SO4, 1.5 mM MgCl2, 0.1% Tween 20, 0.2 mM dNTPs, 0.025 unite/μL Amplicon Taq DNA polymerase, inert red dye, and a stabilizer. The PCR conditions were 5 min at 95°C followed by 35 cycles of 30 s at 94°C, 1 min at 60°C, 2 min at 72°C, and a final elongation of 72°C for 7 min [].The PCR products were separated on a 1.5% agarose gel with 1x TAE buffer and visualized by staining with ethidium bromide.The PCR positive samples were purified using an Accuprep Gel Purification kit (Bioneer, Deajeon, Korea) then sequenced (MWG-Biotech, Ebersberg, Germany) by the primers employed in the PCR. Sequence alignments were constructed using the program CLUSTAL W version 1.83 (http://www.ddbj.nig.ac.jp/search/clustalwe.html). […]

Pipeline specifications

Software tools MUSCLE, Clustal W
Databases DDBJ
Application Nucleotide sequence alignment
Organisms Mus musculus, Rattus norvegicus, Toxoplasma gondii, Rattus rattus, Homo sapiens
Diseases Infection