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Rapid and Quantitative Detection of
Toxoplasma Gondii by PCR
J.M. Costa 1, P. Ernault 1, and S. Bretagne2
Molecular Biology Laboratory, American Hospital of Paris, Neuilly-sur-Seine, France.
2
Laboratoire de Parasitologie, Hospital Henri Mondor, Créteil, France.
1
I
ntroduction
Toxoplasmosis is a worldwide infectious disease
caused by the protozoan parasite, Toxoplasma
gondii (T. gondii). The infection is usually asymptomatic and harmless in immunocompetent
patients, but can be life-threatening or responsible for severe sequelae in immunocompromised
individuals, such as fetuses, HIV-positive, and
transplant patients. In these situations, early treatment significantly reduces the extent of the
damage [1]. However, the classical diagnosis of
toxoplasmosis based on serological tests is inefficient and inadequate in these patients. Therefore,
the diagnosis is based on the direct demonstration
of the parasite in tissues or biological fluids. This
can be achieved by tissue culture or mouse inoculation. However, tissue culture is not very sensitive and inoculation of mice takes more than three
weeks to complete [2]. PCR overcomes these
shortcomings [3]. PCR is sensitive and the diagnosis can be confirmed within one day. The use of
PCR has greatly improved and simplified the “home
brew” applications for prenatal diagnosis of toxoplasmosis, in particular, making it faster, more
sensitive, and decreases morbidity, because it is
currently based on amniocentesis alone [4].
However, the main risk concerns false-positive
results arising from contamination with previously
amplified products. The recent development of the
Ý
Figure 1: Hybridization Probes procedure. Fluorescence is emitted by LC-Red
640, when both Hybridization Probes are annealed to one strand of the amplification
product that is generated during PCR. The PCR fragment size is 126 base pairs.
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BIOCHEMICA · No. 3 n 1999
real-time PCR procedure means that this risk can
now be controlled. We have therefore, tested the
feasibility of this technique in a standard research
laboratory and compared the results with those
obtained in our laboratory, henceforth referred to
as conventional PCR.
M
aterials and methods
Samples
T. gondii DNA (strain RH) was prepared using a
conventional phenol-chloroform procedure, and
purified parasite was obtained from ascites fluid in
inoculated mice. The amount of parasite
(tachyzoite equivalents) was determined by spectrophotometric measurement (one parasite
z 0.1 pg of DNA).
Nucleic acids from amniotic fluids were extracted
using the High Pure PCR Template Preparation Kit
(Roche Molecular Biochemicals), according to the
manufacturer’s recommendations. Five to 20 µl of
the eluate were subjected to PCR amplification.
Primers and probes
The forward (BO3 23-mers) and the antisense (BO4
25-mers) amplification primers were designed to
amplify specifically a 126 bp fragment in a wellconserved region of the tandemly-repeated B1
gene of T. gondii [5]. For the conventional procedure, the sense primer was 5' biotin-labeled to
allow detection and specificity checking of PCR
products using a digoxigenin-labeled probe (see
below). The same set of primers was used in combination with specific hybridization probes, labeled
respectively with LightCycler-(LC)-Red 640 (LC6
25-mers) and fluorescein (LC5 27-mers), for
detection of the PCR products using fluorescence
resonance energy transfer on the LightCycler
Instrument (see Figure 1).
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Conventional PCR
The samples (20 µl) were amplified in a 50 µl
reaction mixture containing: 2.5 mM MgCl2; 50 mM
KCl; 10 mM Tris-HCl, pH 8.3; 0.2 mM each of
dATP, dGTP, and dCTP; 0.4 mM dUTP; 20 pmoles
each of T. gondii primers; 0.5 units of Uracil-DNA
glycosylase (UNG), and 1.25 units of AmpliTaq
Gold (Perkin Elmer). The samples were initially
incubated for 5 min at 50°C to allow the UNG to
act. This incubation was followed by a 10-minute
step at 95°C to denature the DNA and activate the
AmpliTaq Gold. The temperature cycling (42 cycles
at 95°C and 60°C, 30 s in each case, and 72°C for
1 min) was performed in a 48-well thermal cycler
(Perkin Elmer Cetus). Amplification products were
detected by PCR ELISA using digoxigenin-labeled
probes and the PCR ELISA DIG Detection Kit
(Roche Molecular Biochemicals).
Real-time detection
PCR amplification was performed with the
LightCycler DNA Master Hybridization Probes kit
in a standard PCR reaction containing 0.5 µM of
each primer and 0.25 µM of each probe with 5 µl
of sample. A hot start procedure was systematically employed by the addition of an anti-Taq DNA
polymerase (CLONTECH) to the amplification
reaction mixture. Carry-over was systematically
prevented by using heat-labile Uracil-DNA glycosylase (UNG) (Roche Molecular Biochemicals).
The amplification and detection were carried out
in a LightCycler Instrument as follows. The
reaction mixture was initially incubated for 5 min
at room temperature to allow the UNG to act. This
incubation was followed by a 2-minute step at
95°C to denature the DNA and to inactivate both
the UNG and the anti-Taq DNA polymerase.
Amplification was performed for 50 cycles of
denaturation (95°C, 5 s, ramp rate 20°C/s), annealing (60°C, 10 s, ramp rate 20°C/s), and extension
(72°C, 15 s, ramp rate 2°C/s). Fluorescence was
acquired at the end of each annealing phase.
Real-time Positive
PCR
Negative
Figure 2: Comparative sensitivity between real-time PCR and the
conventional procedure. 2a. Screen shot of real-time detection, showing the development of fluorescent signals during PCR. 2b. Schematic presentation of results for the
microtiter plate detection format.
R
esults and discussion
Using purified DNA diluted in a Tris-HCl buffer,
the real-time PCR detected the same amount of
DNA as the conventional PCR. The amount of DNA
equivalent to one tachyzoite was systematically
detected by both methods (Figure 2). The results
obtained with 22 frozen amniotic fluids (10 negative and 12 positive) were identical for both PCR
techniques (Table 1).
Specimen No.
T. gondii quantity
96017
7.96 x 102
96001
1.62 x 103
98067
4.29 x 100
98132
2.90 x 100
98069
0.65 x 100
98026
2.76 x 100
98198
3.98 x 102
98117
2.04 x 101
Conventional PCR
98190
3.55 x 102
Positive
Negative
98191
1.02 x 100
12
0
98211
1.52 x 101
0
10
98115
3.08 x 102
Ý
Table 1: Analysis of amniotic fluids for the
presence of T. gondii by PCR. Comparison of conventional
and real-time PCR results.
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Ý
Ý
Table 2: Quantitative analysis of PCR-positive
samples. Quantities are expressed in number of tachyzoites
per milliliter of amniotic fluid.
BIOCHEMICA · No. 3 n 1999
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Pg06.fm Page 8 Tuesday, October 26, 1999 1:06 PM
Û Figure 3: Real-time quantitative PCR analysis.
3a: Analysis of the amplification plots for the standard
concentrations (in duplicate) of T. Gondii.
3b: Standard curve; plot of the crossing point (cycle number)
against the input target quantity.
A
cknowledgments
For quantitative analysis of the parasite DNA, the
results obtained with the LightCycler Instrument
were compared with the standard curve of T. gondii
DNA (Figure 3). The parasite burden was
extremely variable, ranging from 1.62 x 10 3/ml to
0.65 x 10 0/ml, although most of the parasite burden
was weak (Table 2). Nevertheless, even the weak
parasite burden must be detected as the consequences can be damaging for the fetus.
Our results showed that PCR amplification of the
B1 gene of T. gondii on the LightCycler Instrument
is as sensitive as the conventional PCR used in our
laboratory. Moreover, the LightCycler Instrument
has additional advantages over conventional PCR,
including (i) contamination prevention (the
closed-tube system decreases the carry-over of
PCR products), (ii) speed (results can be obtained in
less than one hour, compared to 5 h with conventional PCR), (iii) the quantitative analysis, which
may have prognostic implications and could thus
prove useful in comparing different drug regimens.
Product
Cat. No.
Pack Size
LightCycler Instrument
2 011 468
1 instrument plus accessories
LightCycler – DNA Master
Hybridization Probes
2 015 102
2 158 825
1 kit (96 reactions)
1 kit (480 reactions)
LightCycler-Red 640 NHS ester
2 015 161
1 vial for $ 250 nmol
oligonucleotides
High Pure PCR Template
Preparation Kit
1 796 828
100 reactions
PCR ELISA (DIG-Detection)
1 636 111
192 detection reactions
Uracil-DNA Glycosylase
(UNG), heat-labile
1 775 367
1 775 375
100 units
500 units
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BIOCHEMICA · No. 3 n 1999
The authors gratefully acknowledge the assistance
of Roche Molecular Biochemicals in the synthesis
and labeling of the LC-Red 640 and fluorescein
hybridization probes. All the presented data were
generated using a LightCycler Instrument supplied
by Roche Molecular Biochemicals, France.
References
[1]Roizen, N., Swisher, C.N., and Stein, M.A. et al. (1995)
Pediatrics, 95, 1: 11-20.
[2]Fircker-Hidalgo, H., Pelloux, H., Muet, F., Racinet, C.,
Bost, M., Goullier-Fleuret, A., and Ambroise-Thomas, P.
(1997) Prenat. Diagn. 17, 9: 831–835.
[3]Bretagne, S., Costa, J.M., Vidaud, M., Tran Van Nhieu,
J., and Fleury-Feith, J., (1995) J. Clin. Microbiol., 33, 6:
1662-1664.
[4]Hohlfeld, P., Daffos, F., Costa, J.M., Thulliez, P.,
Forestier, F., and Vidaud, M., (1993) N. Engl. J. Med.,
331, 11: 695-699.
[5]Burg, J.L., Grover, C.M., Pouletty, P., and Boothroyd,
J.C., (1989) J. Clin. Microbiol., 27, 8: 1787-1792.
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