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Comparative Sensitivity of Fluorescence Resonance
Energy Transfer Quantitative PCR and
Conventional Nested PCR in detection of
Toxoplasma parasiteAmany A Abd El-Aal, Enas Zakaria, Nehal A Hanf,
Asmaa A Abd El-Aal
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Amany Ahmed Abd El-AalProfessor of Parasitology
Faculty of Medicine , cairo University
Presented by:
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Introduction
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Toxoplasma gondii (T. gondii) is a common cause ofinfection in many warm-blooded animals, including
humans. Between 15 and 85% of the world adult
human population is chronically infected with T.
gondii depending on geographical location.
Introduction
Most cases of human infection are mild, but
devastating disease can occur in immune-
compromized individuals and congenitally infected
fetuses that may lead to serious complications. Early
diagnosis is crucial to start treatment that drastically
reduces the extent of the damage.
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Serological diagnosis of active infection is unreliable because
reactivation of hidden infection is not always accompanied by
changes in antibody levels, and evolution of a latent or dormant
toxoplasmosis is highly unpredictable. Moreover, the presence ofIgM does not necessarily indicate recent infection.
Introduction
Application of PCR has evolved as a sensitive, specific, and rapid
method for the detection of T. gondii DNA in different samples,
accordingly serving confirmation of active infection.
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There is a wide variety of protocols used by different Research
laboratories that employ the three-stages conventional PCR
procedure (extraction,amplification and detection). In addition to
a 4th stage in case of semi-quantitative analysis of the specific
genome using gel documentation system and markers with known
molecular weight size .
Introduction
On the other hand, accelerating the molecular diagnosis of
toxoplasmosis by performing real-time PCR protocols has been
reported. This technique allows amplification and simultaneous
detection of DNA in 1 h.Unlike many bacterial and viral infections there is no available
IVD (In Vitro Diagnostic) molecular technique for parasitic
infection.
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Introduction
According to the method by which the primers and probes
hybridize to the target gene, there are different qPCR protocols as
TaqMan probes, Molecular Beacons and fluorescence resonance
energy transfer (FRET) probes.
Concerning qPCR, fluorescently labeled specific probes was
reported as a highly sensitive and specific method of detection,
as only the desired PCR product is detected.
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AimThis work aimed to evaluate sensitivity of 2 different PCR
protocols in detection ofToxoplasma parasite, conventional nested
PCR and Fluorescence Resonance Energy Transfer (FRET) real
time PCR.
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Methodology
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The target DNA of the cloned sample
(Roche Diagnostics) was provided in 6
different quantities to yield from 10copies to 106 copies ofToxoplasma target
molecules once eluted in 5 l PCR-
grade water . Reaction mixture was
added to each sample and was then
mixed by pipetting the solution up anddown 10 times.
Methodology
Toxoplasma samples
T. gondii (P-strain) tachyzoites werecollected by peritoneal lavage of infected
mice. The number of tachyzoites in
suspension was determined by counting
on a haemocytometer prior to DNA
extraction.
Cloned Toxoplasma DNA samples Toxoplasmagondii tachyzoites
Negative control: By replacing the template DNA with water.
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DNA was extracted from Toxoplasma
tachyzoites using fully automated
MagnaPure Compact Instrument . Final
pellets were suspended in 30L of TE buffer
and stored
at 70C until used.
DNA extractionMethodology
The amount of parasite genome (tachyzoite
equivalents) was determined by
spectrophotometer (one parasite ~ 0.1 pg of
DNA). Accordingly different dilutions were
prepared
Perform 116 nucleic acid purifications from the sample in 15-40 minutes
The system is highly flexible concerning the sample and elution volume needed
to obtain the necessary nucleic acid concentrations for application.
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Nested Polymerase chain reaction:
Methodology
The fully automated thermo-
cycler (Q cycler Quanta
Biotech).
Nested primer sets targeted B1
gene were;
The outer bases were from171 to
190 and from bases 602 to 583
producing an amplified product of
432 bp.
Inner primers were from bases 180 to 196 and from bases 392 to
372 producing an amplified product of 213 bp.
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The amplification products weredetected by gel electrophoresis
using 3% agarose gel in 1 tris-
borate-EDTA buffer.
DNA bands were visualized using
0.5% ethidium bromide in thepresence of ultraviolet light.
Methodology
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PCR, targeting T. gondii 134 bp
repeated region (more than 200-
fold) was amplified with specific
primers and Fast-StartDNA
MasterPLUS Hybridization
Probes Kit. The resulting PCR
fragment ofT. gondii was analyzed
with a probe labeled withLightCyclerRed 640 (detected in
channel 640).
Methodology
QuantitativeFRET-PCR(Fluorescence Resonance Energy Transfer)
protocol:
The LightCycler Real-Time PCR
System with its screen showing the
soft war
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In this technique, two labeled oligonucleotide probes that bind to
the PCR product in a head-to-tail fashion
When the two probes bind, their
fluorophores come into close,
allowing Energy Transfer froma donor to an acceptor
fluorophore.
Therefore, fluorescence is
detected during the annealingphase of PCR and is proportional
to the amount of PCR product.
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Prior to the performance, a standard curve was automatically
drown, using known samples with known genomic quantities to
facilitate estimation of genomic quantites of the unknown
samples.
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Optimization of qPCR was done to avoid non specific
reactions (primer dimmer ).
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Results
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Real-time PCR detected the same amount of DNA extracted from
different dilutions of Toxoplasma tachyzoites as the conventional
PCR. The amount of DNA equivalent to one tachyzoite wassystematically detected by both methods.
Results
Using the cloned Toxoplasma DNA, all concentrations from 101 to
10 6 were detected by qPCR, while nPCR failed to detect the
lowest concentration (101).
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Nested PCR was sensitive to detect102 copies while FRET assay
showed a sensitivity of 101 copy.
Results
Crossing points (Cps) showed different values ranging from 39.68
to 12.88 reflecting the different DNA quantities (from 1.7x 101 to9x 1010) .
Quantitative genomic estimation of these positive samples inqPCR was ranging from 1.7x 101 to 9x 1010.
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In addition of acurate Quantitative genomic estimation of these positive
samples, qPCR technique can simultaneously identify genotype of the
target genome using reference strain
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1
2-5
6
Positive samples in relation to the negative control . First signal at
cycle number 13 followed by 5 signals starting from cycle 17 to
22 . The last signal is a weak one at cycle 30.
Amplification product becomes detectable
within the baseline setting of cycles 13 to cycle 30
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Results
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Conclusions
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Conclusion
The main advantages of Real-time PCR
over nested-PCR assays as follow; (i) it isfar less labor-intensive (only one PCR step,
compared to at least two in nested-PCR);
(ii) qPCR is performed in a closed systemwhere post-PCR handling is not required
(i.e., transfer of amplified template from the
primary to the secondary amplification
reaction and agarose gel electrophoresis for
the detection of PCR products; thisconstitutes a major advantage since the risks
of contamination are minimal);
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As for all parasitic diseases, the PCR diagnosis of toxoplasmosis isnot standardized. Due to the previously mentioned advantages of qPCR
over conventional PCR, It seems highly probable that there will be an
agreement on Real-time PCR assays in the next few years. However, the
agreement on the best sequence to be amplified will be more difficult.
Conclusion
For a possible application of Real-time PCRs in routine diagnosis,
protocols should be optimized and carefully evaluated in a larger
number of clinical samples before they are implemented as routinediagnostic method for toxoplasmosis.
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Thanks