Download PCR applications in diagnosis of parasitic diseases

By
Dr. Zainab khalid
Introduction
to PCR
PCR was invented in
1984 by ( Kary mullis
) & he received the
Nobel Prize in
chemistry in 1993, for
his invention
What is PCR?
PCR is an exponentially progressing
synthesis of the defined target DNA
sequences in vitro to get millions of
copies.
Why “Polymerase”?
It is called “polymerase” because the
only enzyme used in this reaction is
DNA polymerase.
PCR tube
THERMOCYCLER
PCR principle :
It allow asingle DNA sequence to be copied (million of
times).
PCR of two main types:
RT_PCR for amplification of RNA.
RT_PCR quantitative measurement of RNA or DNA:
_The amplification of DNA occurs by repeated cycles of 3
different temp. depended steps:
1-Denaturation:separation of the two strands of DNA by
heating up to 94c for 2 min. without breaking the major
bonds of its chain.Producing single stranded DNA
template
For replication by thermostable DNA polymerase.

 2-Anneling: In this step Primers are attached to the
single DNAstrand template at their complementary regions
, in this step temperature is reduced to 40-60c.
3-Extention: the DNA polymerase synthesize
complementary strand. The enzyme reads the
opposing strand sequence & extends the primers by
adding nucleotides. The temp. in the range 70-74c
Denature (heat to
94oC)
Anneling-Lower
temperature to 40-60oC
Extention-Increase
temperature to70-74oC
DNA polymerase
DNA copies vs Cycle number
2500000
DNA copies
2000000
1500000
1000000
500000
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Cycle number
15
16
17
18
19
20
21
22
23
Amplifies target sequences & increases
sensitivity.
1. Ribosomal DNA/RNA.

Highly sensitive.
2. Specific sequences of genomic DNA.

Highly specific for single species .
3. Random primer amplification

Very highly sensitive .
Advantages:
Small amount of the sample.
Very sensitive .
+ rapid & time consuming test.
Disadvantages:
Expensive .
PCR can fail: - Contamination & false positives.
Can not differentiate between living & dead
parasite since PCR detect DNA that may still in the
body even after treatment & elimination of the
parasite.
 RT_PCR quantitative measurement of RNA or
DNA:
 there will be detection of “amplification associated
florescence” in each cycle. It is used in diagnosis of
many conditions other than parasitological
infections.
Parasites can be diagnosed by PCR :
1- Malaria :
- Amplification of oligonucleotide
primer pairs within the ribosomal
RNA genes which is specific to each
species by the Polymerase Chain
Reaction (PCR), to detect each malaria
species.
Traditional diagnosis of Malaria
2- Leishmania donovani
 Amplification of xeno-mini gene found
uniqu on it,s genome in bone morrow
or tissue sample.
3- Toxoplasma gondii
 Toxoplasma gondii has a three (I, II, and III) strains
according to genetic structure . Type II strains
cause most cases of symptomatic human
infections.This strains can be detected by
amplification of DNAof the parasite in blood
sample or amniotic fluid by PCR.
4-Trypanosoma cruzi
 Dgnosis of acute infection with
Trypanosoma cruzi, the protozoan parasite
that causes Chagas disease by amplification
of DNA in blood sample give earlear
diagnosis than traditional methods by 3-9
days .
3- E. histolytica/E. dispar, G. lamblia and C.
parvum/C. hominis infections
Their diagnostis by PCR is very limited
by
*time-consuming methods for the
isolation of DNA from faecal
specimens
* the presence of inhibitory substances
in such samples.
But it is usful in the epidemiological studies
to collect data on the prevalence of E.
histolytica and E. dispar which is more
advantageous than the ELISA by:
 time-consuming
 sensitive
 Can detect five cysts in the stool sample
 can be performed in one day and selectively
differentiates E. histolytica from E. dispar
DNA.
Conclusion:
The speed and ease
sensitivity & specificity of PCR
has made PCR the most widely used
and powerful technique with great
spectrum of research and
diagnostic applications.