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 (CT) indicative of the quantity of the target gene were determined. Typically, a CT 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 CT 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.

A prospective study of diagnosis of Toxoplasma gondii infection after bone marrow transplantation

“…This DNA element is repeated 35 times in the genome of the parasite, and the number of repeats provides optimal detection of T. gondii DNA by PCR (18). Recently, a 529 bp element that is repeated 200–300 times in the genome of T. gondii has been described (9, 19–23). It has been proposed that a PCR target containing more repeats than the B1 gene may increase the analytical sensitivity (24–28).…”

Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis

“…A variety of real-time PCR assays and PCR targets have been developed for the rapid and sensitive detection of T. gondii [4][5][6], including the B1 gene, which occurs in 35 copies in the parasite genome [7][8][9][10][11][12][13]. A further repeat element of unknown function, which occurs in 200-300 copies, has recently been described in T. gondii [14].…”

Development of a multiple internal control for clinical diagnostic real-time amplification assays

“…The oligonucleotide sequences, used for DNA real-time amplifications, are complementary to the published sequences of the C, VP1, BALF5, B1 and pp150 genes of HBV, huPoV, EBV, Toxoplasma gondii and CMV, respectively (Tables 1 and 2) (Kimura et al, 1999;Biel et al, 2000;Lin et al, 2000;Marchant et al, 2003;Leb et al, 2004;Stöcher et al, 2004). To allow distinction between the fluorescence signals of the pathogen and mIAC, the pathogen-specific probes were labelled with the fluorescent reporter dye 6carboxyfluorescein (FAM) at the 5 0 end and the quencher dye 6-carboxytetramethylrhodamine (TAMRA) at the 3 0 end, whereas the probe specific for mIAC was labelled with the fluorescent reporter dye 6-carboxy-4 0 ,5 0 -dichloro-2 0 ,7 0dimethoxyfluorescein (JOE) at the 5 0 end and the quencher dye TAMRA at the 3 0 end ( Table 2).…”

“…The majority of reported methods for the detection of hepatitis B virus (HBV), human polyomavirus (huPoV), Epstein-Barr virus (EBV), Toxoplasma gondii and cytomegalovirus (CMV) using real-time PCR methods are directed towards target conserved regions of the C, VP1, BALF5, B1 and pp150 genes, respectively (Kimura et al, 1999;Biel et al, 2000;Lin et al, 2000;Marchant et al, 2003;Leb et al, 2004). However, only a few are internally controlled.…”