2015 ACVIM Forum Research Abstract Program

The optimization of a published SYBR Green based quantitative PCR (qPCR) of a fragment of the internal transcribed spacer 2 (ITS-2) gene of C. cayetanensis followed by melting curve analysis were performed as published. To determine the linear range of the reaction, a 10-fold serial dilution of the synthetized oligonucleotide (AF301386) was performed and regression analysis was performed on the resulting standard curve using the instrument software. Intra and interassay variability were determined by running five replicates of the dilution series in three different runs. Specificity for C. cayentanensis was determined by including genomic DNA from G. duodenalis, C. parvum, and Toxoplasma gondii in duplicate along with C. cayentanesis DNA. Optimal annealing temperature was determined by performing an annealing gradient ranging from 53°C to 60°C.