toxo comparative sensitivit 10=10

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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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toxo comparative sensitivit 10=10

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