A quantitative competitive PCR method to determine the parasite load in the brain of Toxoplasma gondii -infected mice

Abstract 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 m...

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Bibliographic Details
Published in:Parasitology international 2008-09, Vol.57 (3), p.347-353
Main Authors: Piña-Vázquez, Carolina, Saavedra, Rafael, Hérion, Pascal
Format: Article
Language:English
Subjects:
Animals
Brain
Brain - parasitology
DNA Primers
DNA, Protozoan - analysis
DNA, Protozoan - isolation & purification
Female
Gastroenterology and Hepatology
Infectious Disease
Mice
Parasite load
Polymerase Chain Reaction - methods
Protozoan Proteins - genetics
Quantitative competitive PCR
Reference Standards
Reproducibility of Results
Sensitivity and Specificity
Toxoplasma - genetics
Toxoplasma - isolation & purification
Toxoplasma gondii
Toxoplasmosis
Toxoplasmosis, Animal - parasitology
Toxoplasmosis, Cerebral - parasitology
Vaccine
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Summary:Abstract 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.
ISSN:1383-5769
1873-0329
DOI:10.1016/j.parint.2008.03.001