Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 5, 2001
Publication Date: June 1, 2001
Citation: Jauregui, L.H., Higgins, J., Zarlenga, D., Dubey, J.P., and Lunney, J.K. 2001. Development of a real-time PCR assay for detection of Toxoplasma gondii in pig and mouse tissues. J. Clin. Microbiol. 39:2065-2071. Interpretive Summary: Outbreaks of human disease from swine products negatively reflect on pork as a safe source of nutritious edible tissue and lowers export potential. Swine have 2 prominent foodborne parasitic disease agents: the muscle worm, Trichinella spiralis,and the tissue protozoan parasite. Toxoplasma gondii. Management changes have virtually eradicated T. spiralis from commercial pork products. However, for T. gondii, changes have reduced but not completely eliminated this foodborne parasite. Therefore tests are needed to determine whether food products are contaminated with this parasite. This manuscript presents data on the development of a sensitive test for T. gondii using the TaqMan assay system. This "Toxo TaqMan" assay covers a broad dynamic range, detecting parasite DNA derived from 1 to 1 million parasites. It is a quick 2-day assay that should be available to diagnostic labs once refinements have been incorporated. This test should help producers to detect parasite contamination and to devise better maintenance to prevent new infections. Since no drugs are available to kill tissue T. gondii parasites, it is essential that producers have improved tools to determine foodborne disease burden. A patent has been submitted for this Toxo TaqMan assay.
Technical Abstract: A highly sensitive and specific method has been developed to reproducibly detect and quantitate Toxoplasma gondii burden in animal tissue samples using 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. gondii and other common protozoa, as well as host animal tissue. This Toxo TaqMan assay was able to detect as little as 0.1 pg of T. gondii genomic DNA, which is equivalent to 1 T. gondii bradyzoite, and has a dynamic range of detection over 6-7 log of T. gondii DNA concenration (from 100 ng to 0.1 pg). Tissues from T gondii 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 with T. gondii parasite concentrations, ranging from 4 parasites to 3.7x105 parasites per gram of spiked pig tissue, with ecellent linearity (RZi parasite concentrations, ranging from 4 parasites to 3.7x105 parasites per gram of spiked pig tissue, with excellent linearity (R2= 0.9776). All T. gondii-infected animals were correctly identified using this techique. Results indicate this assay is applicable to testing swine carcasses and commercial pig products, is compatible with automation technology for potential slaughterhouse usage, and will enable scientists to diagnose and quantitate T. gondii in animal tissues.