Development of a Real-Time PCR Assay for Detection ofToxoplasma gondiiin Pig and Mouse Tissues

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Abstract
A highly sensitive and specific method has been developed to reproducibly detect and quantitateToxoplasma gondiiburden in animal tissue samples usingT. gondiiITS1-derived primers and a fluorogenic probe via real-time PCR. Assay specificity was confirmed against a panel of DNA samples fromT. gondiiand other common protozoa as well as host animal tissue. This Toxo TaqMan assay was able to detect as little as 0.1 pg ofT. gondiigenomic DNA, which is equivalent to 1T. gondiibradyzoite, and has a dynamic range of detection of from 100 ng to 100 fg ofT. gondiiDNA. Tissues from experimentally infected mice and pigs as well as bradyzoite-spiked pig muscle samples were used to test and standardize this technique. Positive signals were obtained withT. gondiiparasite concentrations ranging from 4 to 3.7 × 105parasites per g of spiked pig tissue, with excellent linearity (R2= 0.9776). AllT. gondii-infected animals were correctly identified by this technique. Results indicate that this assay is applicable to swine carcasses and commercial pig products, is compatible with automation technology for potential slaughterhouse use, and will enable scientists to diagnose and quantitateT. gondiiin animal tissues.