Real-time PCR for quantitative detection of Toxoplasma gondii

The protozoan Toxoplasma gondii is one of the most common infectious pathog enic parasites and can cause severe medical complications in infants and im munocompromised individuals. We report here the development of a real-time PCR-based assay for the detection of T. gondii. Oligonucleotide primers and a fluorescence-labeled TaqMan probe were designed to amplify the T. gondii B1 gene. After 40 PCR cycles, the cycle threshold values (C-T) indicative of the quantity of the target gene were determined. Typically, a C-T of 25. 09 was obtained with DNA from 500 tachyzoites of the T. gondii RH strain. T he intra-assay coefficients of variation ICV) were 0.4, 0.16, 0.24, and 0.7 9% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimiz ation of assay conditions, we were able to obtain a standard curve with a l inear range (correlation coefficient = 0.9988) across at least 6 logs of DN A concentration. Hence, we were able to quantitatively detect as little as 0.05 T. gondii tachyzoite in an assay. When tested with 30 paraffin-embedde d fetal tissue sections, 10 sections (33%) showed a C-T of <40 and were sco red as positive for this test. These results were consistent with those obt ained through our nested-PCR control experiments. We have developed a rapid , sensitive, and quantitative real-time PCR for detection of T. gondii. The advantages of this technique for the diagnosis of toxoplasmosis in a clini cal laboratory are discussed.