Real-time PCR for quantitative detection of Toxoplasma gondii

Abstract

The protozoan Toxoplasma gondii is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised 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. The intra-assay coefficients of variation (CV) were 0.4, 0.16, 0.24, and 0.79% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimization of assay conditions, we were able to obtain a standard curve with a linear range (correlation coefficient = 0.9988) across at least 6 logs of DNA concentration. Hence, we were able to quantitatively detect as little as 0.05 T. gondii tachyzoite in an assay. When tested with 30 paraffin-embedded fetal tissue sections, 10 sections (33%) showed a C(T) of <40 and were scored as positive for this test. These results were consistent with those obtained 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 clinical laboratory are discussed.

FIG. 1

Design of real-time PCR for detection of the T. gondii B1 gene. The relative positions of the primers and TaqMan probe in the B1 gene for real-time PCR and nested PCR are shown.

FIG. 2

Real-time PCR detection of the T. gondii B1 gene. (A) Typical amplification plot with 500 tachyzoites as the initial DNA template. Rn, fluorescent signal. (B) The PCR product from panel A was fractionated on a 2% agarose gel followed by visualization with ethidium bromide staining. Lane 1, DNA molecular weight marker; lane 2, 500 tachyzoites; lane 3, no-template control.

FIG. 3

Establishment of the standard curve for quantification of T. gondii. Serial dilutions of T. gondii DNA, ranging from 5,000 to 0.05 tachyzoites, were used as the template for real-time PCR analyses. (A) C_T values were plotted against log (amount of tachyzoites). (B) C_T values for all data points. Similar results were obtained in three independent experiments. NTC, no-template control.