Technique / Molecular Biology / PCR / Competitive PCR

Efficacy of vaccine candidates against toxoplasmosis may be expressed in terms of reduction in cyst number in brains of animals vaccinated and then challenged with a cyst-forming strain of Toxoplasma gondii, compared to non-vaccinated animals. Cyst number generally has been determined by microscopic examination of brain homogenate samples, a technique which has a low sensitivity and is time-consuming. Here we describe a quantitative competitive PCR method, which allows quantifying T. gondii DNA in brain samples. The method uses a primer pair, which allows the amplification of a 301 bp fragment of the 35-fold repeated T. gondii B1 gene and an internal standard (non-homologous competitor) derived from phage lambda, which can be amplified using the same primers and whose size and G/C content are similar to that of the B1 target sequence. The method is sensitive (as few as 10 parasites can be quantified), reproducible, and is not affected by the presence of DNA extracted from mouse brain by means of a simple and rapid technique. It is suitable to quantify the parasite load in the brain of infected mice and to evaluate efficacy of toxoplasmosis vaccine candidates.