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Title: EVALUATION OF A FLUOROGENIC REAL-TIME PCR ASSAY FOR THE DETETION OF TOXOPLASMA GONDII IN BIOLOGICAL SAMPLES

Author
item JAUREGUI, L - BUENOS AIRES
item Higgins, James
item Zarlenga, Dante
item Lunney, Joan

Submitted to: American Association of Veterinary Parasitologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 7/23/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Prevention of Toxoplasma gondii (Tg) infection in swine is a food safety priority for pork producers, since Tg-contaminated pork products can carry serious consequences for human health. In this presentation, the development of a fluorogenic 5' nuclease assay for detection of Tg in pig samples is described. Briefly, the method measures PCR product accumulation nthrough the use of a dual-labeled fluorogenic probe (TaqManTM). Specificit of the assay system was tested by analyzing reactivity on DNA from Tg and other closely related Apicomplexa parasites (Neospora. caninum, Hammondia hammondi, Sarcocystis spp, Eimeria spp., Cryptosporidium parvum) and from host tissues, pigs and mice. Only Tg DNA showed a positive signal. The assay sensitivity was evaluated by testing serial dilutions of Tg DNA (10 ng to 1 fg). Positive signals at 100 fg (~1 parasite) were reproducibly detected, giving assay CT values ~41. Amplification showed linearity over a awide range of Tg DNA concentrations (8 logs). To test assay sensitivity in pork samples, normal pig muscle samples (1 and 10 g) were spiked with different numbers of purified bradyzoites (from 3.7 x105 to 3.7), digested with pepsin-HCl; the DNA was extracted and then assayed at a fixed amount (500 ng total DNA/tube). Positive signals were obtained at 3.7 bradyzoites/g, but with CT values ~43, suggesting the presence of inhibitory factors for the PCR reaction. Comparison of the TaqMan system with conventional tests like mouse bioassay and serology are in process, as well as the use of different target genes (B1 or rDNA). This new technique appears to be a very sensitive and specific assay for Tg infection of pork products.