method for quantifying and assessing the viability of neospora caninum in samples of bovine semen

摘要

Procedure for the quantification of N. caninum in bovine semen samples.; The presence of N. caninum in bovine semen samples is quantified by quantitative real - time PCR, which uses the SYBR Green I. System For the extraction of parasite DNA semen, seminal fluid cell fraction is separated and DNA from each of the fractions using a commercial test is extracted. If frozen sperm diluent, the sample is passed through a chromatography column Sephacryl S-400 and the DNA precipitated with isopropanol and glycogen. The quantification of N. caninum is carried out by interpolating the Ct (threshold cycle: cycle in which a sample is considered positive) on a standard curve constructed with known concentrations of parasite DNA against their respective Ct. For quantification of the host DNA is carried out with another standard curve with known concentrations of genomic DNA.