Development of a real-time PCR assay for detection of Toxoplasma gondii inpig and mouse tissues

A highly sensitive and specific method has been developed to reproducibly d etect and quantitate Toxoplasma gondii burden in animal tissue samples usin g T. gondii ITS1-derived primers and a fluorogenic probe via real-time PCR. Assay specificity was confirmed against a panel of DNA samples from T. gon dii and other common protozoa as well as host animal tissue. This Toxo TaqM an assay was able to detect as little as 0.1 pg of T. gondii genomic DNA, w hich is equivalent to 1 T. gondii bradyzoite, and has a dynamic range of de tection of from 100 ng to 100 fg of T. gondii DNA. Tissues from experimenta lly infected mice and pigs as well as bradyzoite-spiked pig muscle samples were used to test and standardize this technique. Positive signals were obt ained with T. gondii parasite concentrations ranging from 4 to 3.7 x 10(5) parasites per g of spiked pig tissue, with excellent linearity (R-2 = 0.977 6). All T. gondii-infected animals were correctly identified by this techni que. Results indicate that this assay is applicable to swine carcasses and commercial pig products, is compatible,vith automation technology for poten tial slaughterhouse use, and will enable scientists to diagnose and quantit ate T. gondii in animal tissues.