Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity.Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii.We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.Toxoplasmosis is an infectious disease caused by the protozoan Toxoplasma gondii effecting individuals throughout the world. Two main subpopulations are highly susceptible to this parasite: the fetus and an immunocompromised individual. Congenital infection may induce spontaneous abortion or serious sequelae when maternal infection occurs during pregnancy. Both the damage for the fetus (the sooner, the more deleterious) and the frequency of trans-placental transmission (the later, the more frequent) is usually depending on the stage of gestation. In the immunocompromised host, the prognosis for cerebral disseminated toxoplasmosis is poor, with a mortality rate of 63% [1]. For these two sub