The Polymerase Chain

Reaction (PCR)
the Plasmodium malaria
 Experiment Goals
• Understand how PCR technique works
• Perform PCR experiment
• Analyze PCR products
 What is PCR?
Definition

 The polymerase chain reaction (PCR) is a technique to amplify a

piece specific of DNA very rapidly outside a living cell.

 Tehnik sintesis & amplifikasi DNA spesifik secara in Vitro.

 Development of PCR
 1971: Khorana described basic principle
of DNA replication using DNA primers
 1983: Dr. Karry Mullis developed PCR
technique, for which he received the
Nobel Prize in Chemistry in 1993.
 Malaria Problems

 Vector control is the key strategy

 Resistance of the DDT & drugs (ACT)

 No vaccine for malaria yet

 Limited Global efforts to

control/eliminate/eradicate malaria
 Rising of Zoonosis Plasmodium knowlesi
Lab Methods for Malaria Parasite Detection
and Exposure

Visualize Parasite Case Management

Morphology
(Microscopy)

Detect parasite Case Management

Products (RDTs) Since 2014
(RDT QA in Indonesia)

Detect nucleic acids Research and selected

(Molecular) applications

Detect host biomolecules Past and recent exposure

Antibodies/antigens (IFA, ELISA, Luminex and
metabolites others) Surveillance
 Diagnostic Tools
Characteristics and Limitations
Microscopy
 Sensitivity range 50-200p/uL (expert LOD=20 p/ul)
 Highly important to provide training and quality management
(P. knowlesi diagnosis is challenging)
RDTs
 Sensitivity ~100p/uL
 hrp-2 gene deletion (false negative test results)
 No P. knowlesi specific RDT available
Molecular tools
 Recommended for detection of submicroscopic parasites
 Sensitivity varies and WHO recommends 2 p/ul
Rapid Diagnostic Tests (RDTs) & Malaria Diagnosis

RDTs are antigen-capture based assays detecting in whole blood:

1. P. falciparum Hisitidine-Rich Protein-2 (HRP-2)
2. Plasmodium lactate dehydrogenase (p-LDH)
3. Plasmodium aldolase enzyme

HRP-2 is a stable soluble exported protein composed of largely histidine, alaline and
aspartic acid amino acids arranged in a variety of tandem repeats.

Ex

10
Sensitivity and Specificity among Different Plasmodium
Blood samples from persons non-infected or mono-infected
with one of four human malarias

Rogier, et al, Under Review

1. HRP-2 signal only present for P. falciparum infections.

2. Limit of detection for any of the three antigens is ~1 parasite/uL
blood.
3. Different combinations of antigen positivity indicate speciation and
infection characteristics.
Center for Global Health

Division of Parasitic Diseases and Malaria

 Use Existing Tools for Surveillance but Next Generation
RDTs Are Needed

P. falciparum
 HRP-2 gene deletion (false negative test results)
 (South American Amazon and Eriteria)
 Timika, Papua (Report from Eijkman Institute ±18%)

P. vivax
 Improve sensitivity and specificity

P. knowlesi
 No RDT available for species specific diagnosis-highly needed
in Southeast Asia
Molecular tools for P. knowlesi
 The Distribution of Plasmodium knowlesi 2017-2019 (115 Cases)
Prov. Aceh 78 Cases
Prov. Kaltim
1 kasus
Prov. Sumbar 3 Cases
Prov. Kalteng
5 kasus

Prov. Sumut 2 Cases

Prov. Kalsel 20 Cases

Prov. Jambi 1 Cases

Sumber: PBTDK-MoH, Republic Indonesia, 2019

 Anamnesis (Lapangan): 3-7 hari
1.Demografi
2.Gelaja dan keluhan utama
3.Dilengkapi waktu dan tanggal
pengambilan sampel

Slide + Kertas saring→Lab. Parasitologi (3-5 hari)

Mikroskopis (cross check 3 expert) jam

Positif (+)
Negatif (-)

1. Mix infection-PCR (Semi-Multiplex) PCR secara pooling

2. Single (Nested PCR) (1-2 hari)
3. Kontrol (+/-)
4. 1-2 hari

Gold Kriteria:
FEEDBACK 1.Demam & Riwayat demam
(non RESMI & RESMI) 2.Riwayat kontak Macaca
5-7 hari 3.Tinggal/bekerja di hutan
 Permasalahan Sampel Yang Tiba di Lab.
Parasitologi-PBTDK

Giemsa TERLALU PEKAT

Sampel LENGKET pada
Pengeringan tidak sempurna Sampel darah TIDAK ADA ID
TERLALU SEDIKIT Filter Paper
Permasalahan Sampel (ID yang tidak Jelas)

Nama Yg Tidak Jelas Tidak ada Keterangan

Jenis Kelamin
Genus DNA Target

Sumber: Singh, et al. Clin Microbiol Rev, 2013

Species DNA Target

Sumber: Singh, et al. Clin Microbiol Rev, 2013

Sekuens Primer Plasmodium knowlesi

Source: Imwong et al., 2009, J. Clin. Microbiol

Molecular Tools are Critical for Reference
Lab Investigations and in Special
Situations
Molecular diagnostics can help to confirm species identification
and reference lab test
(P. knowlesi identification, mixed species infection)

Elimination settings (confirm species)-certification

Some PCR tests for P. knowlesi detection can cross react with
other species of malaria parasites (P. vivax)

Our attempt to develop new P. knowlesi specific PCR test

PLoS One, Feb 2012, Vol 7, e31848
Center for Global Health

Division of Parasitic Diseases and Malaria

Commonly used 18S rRNA gene based PCR cross-reacts with other
human and primate malaria species

23

Lucchi et al., PLoS One, Feb 2012, Vol 7, e31848

A gene target (Pkr 140-5) from P. knowlesi specific genome identified for
a species specific molecular diagnostic test

Lucchi et al., PLoS One, Feb 2012, Vol 7, e31848 24

Specificity of the P. knowlesi primers tested against five different simian-
infecting malaria parasites

Primer Pkr140-3

Primer Pkr140-4

Primer Pkr140-5

25

Lucchi et al., PLoS One, Feb 2012, Vol 7, e31848

A gene target (Pkr 140-5) from P. knowlesi does not cross react with
several strains of P. vivax including one from Indonesis

Lucchi et al., PLoS One, Feb 2012, Vol 7, e31848 26

 The Protocols of Samples Analysis (US-
CDC)
Assessment Protocol used Laboratory Location

PCR kit Promega, Madison, USA, Cat. No. #A3511

Qiagen, Blood Mini Kit, 250, Cat, No. 51106

Isolasi DNA (50-75 µL) USA. Cat. No. A-1123

Agarose (2%) Promega, Madison, USA. Cat. No. 2022-01-12

NIHRD Lab-Minisrty
of Health
ETBR (3 µL) Invitrogen, Cat. 155585
Ladder (3 µL) Geneaid, 100 bp, Cat. No DL.004
Elektrophoresis Bio-Rad/80 volt/60 minutes

Length target 200 bp

amplification

PCR Parameters (35 x) 95° C-2.0’/94° C-30”/57° C-30”/72° C-30”/ 72°

C-5.0’ and 4° C-∞
PCR Mix (US-CDC)
One Step PCR Plasmodium knowlesi Test-1
One Step PCR Plasmodium knowlesi Test-2
Eijkman Test
Nested-2 Temp Annealing Test (Singh et al., 2013)
 Temperature Annealing Test
(Single Step PCR US-CDC Method, Naomi et al., 2012)

Note:
1.DNA= 2 ng/mL (slide)/Vol= 2
ul/PCR reaction
2.Ladder 3 µL (Invitrogen)
3.Agarose 2 % (Invitrogen)
4.El-fo= 80 volt/70 mnt
5.K (+) = SB-001
6.P.f/P.v/P.m/P.o (US-CDC)
7.Annealing 59°C (32 cycle)
8.Primer 2 µL
Single Step PCR US-CDC Method, Naomi et al., 2012)

Note:
1.DNA= 0.2 ng/mL (slide)
2.Ladder 3 µL (Invitrogen)
3.Agarose 2 % (Invitrogen)
4.El-fo= 80 volt/70 mnt
5.K (+) = SB-001
6.P.f/P.v/P.m/P.o (US-CDC)
7.Annealing 59°C (32 cycle)
8.Primer 2 µL
 Commonly used PCR methods P. knowlesi detection

No Target gene LOD Publications

1. SSU rRNA 1-6 parasites/ul Singh et al. 2004

2. SSU rRNA 1-10 parasites/ul Imwong et al. 2009

3. SSU rRNA 1-6 parasites/ul Lee et al. 2011

4. unidentified 1 parasites/ul Lucchi et al. 2012

5. SICAvar 0.1 parasite/ul Lubis et al. 2017

LOD= limit of detection

 Result of PCR test (n= 11) Badan Litbangkes

Isolate Lee (Singh) CDC (Single step) Imwong SICAvar Result

Aceh (Sabang & Barat & Selatan) √ √ √ Not tested +++
Sumbar √ X √ Not tested ++
Sumut √/x X X Not tested +
Kaltim √ √ √ Not tested +++
Kalteng √/x √ - Not tested ++
Kalsel √/x X - Not tested ++
Jambi Samples N/A Samples N/A Samples N/A Not tested -
 PCR results of P. knowlesi (source: CDC, 1.47 x 10E-1 ng/ul)

PCR methods X 10E-1 X 10E-2 X 10E-3 X 10E-4 X 10E-5 X 10E-6 X 10E-7 Sensitivity
ng/ul ng/ul ng/ul ng/ul ng/ul ng/ul ng/ul Rank

Lucchi et al. Pk Pk neg neg neg ND ND 4

Imwong et al. Pk Pk Pk Pk neg ND ND 3

Lubis et al. Pk Pk Pk Pk Pk neg ND 2

Real time Pk Pk Pk Pk Pk Pk neg 1
PCR
 Tugas Lab. Parasitologi PBTDK (2 mg
kedepan)

1. Uji coba ulang bersama Eijkman

a.Penyamaan protocol single step PCR-US-
CDC
b.Konsentrasi Primer dan DNA
c.Sampel yang di gunakan representative
dari Indonesia (blind test follow)
d.Sequencing
2. Plasmodium bank
3. PfK-13 (artemisinin resistance)
 PCR Applications
• PCR is now a common and often indispensable
technique used in medical and biological research labs
for a variety of applications.

•Structural analysis •Mapping

•DNA typing •Site-directed mutagenesis
•Disease detection •Sequencing
•Cloning •Forensic medicine
•Mutation analysis •Scientific research
•Detection of gene •Pre-natal diagnosis
expression
 How does PCR work?
1. DNA is denatured (H-bonds are broken
between strands of DNA with heat), 94oC
2. Primers attach to complementary sequences
of single stranded DNA, 55-60oC
3. DNA polymerase attaches to primer with
ssDNA and extends DNA fragment, 72oC
4. Thus, making double stranded DNA
5. This is done by changing the temperature
 PCR Reaction Components

1) Target DNA - contains the sequence to be amplified.

2) Pair of Primers - oligonucleotides that define the sequence
to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.

4) Thermostable DNA Polymerase - enzyme that

catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme

6) Buffer solution – maintains pH and ionic strength of

the reaction solution suitable for the activity of the
enzyme
 1) Target DNA
Target of DNA (Sebagai cetakan untuk
pembentukan molekul DNA baru)
1.a single gene
2.part of a gene
3.or a non-coding sequence
 DNA Quality
DNA should be intact and free of
contaminants that inhibit amplification.

1. Contaminants can be purified from the original

DNA source (Heme from blood, and melanin from
hair).

2. Contaminants can be introduced during the

purification process (Phenol, ethanol, sodium
dodecyl sulfate (SDS) and other detergents, and
salts).
 DNA quantity
More template is not necessarily better.

1.Too much template can cause

nonspecific amplification.

2.Too little template will result in little or

no PCR product.
 How Big A Target is?
1. Amplification products are typically in the size
range 100-1500 bp.

2. Longer targets are amplifiable >25 kb.

3. Requires modified reaction buffer, cocktails of

polymerases, and longer extension times.
 2) Pair of Primers
1. Primers define the DNA sequence to be
amplified—they give the PCR specificity.
2. Primers bind (anneal) to the DNA template
and act as starting points for the DNA
polymerase (DNA polymerases cannot
initiate DNA synthesis without a primer).
3. The distance between the two primers
determines the length of the newly
synthesized DNA molecules.
 Primer
1. Sebagai pembatas fragmen DNA target
yang akan di copy.
2. Menyediakan gugus Hidroksi (-OH)
pada ujung 3’ (ekstensi DNA)
 Hal yang diperhatikan dalam Primer

1. Panjang primer (18-30 bp) Mispriming

2. Komposisi primer
a. % G&C primer =/>G&C target
b. Melting temperature (TM) [2 x (A+T) + 4 x
(G+C)].
c. Tm yang baik 50-65° C
 3) dNTPs
(deoxy nucleotide triphosphates)

1. The building blocks for the newly

synthesized DNA strands.
2. dATP, dGTP, dCTP or dTTP
dNTPs, MgCL2, Polimerase DNA
1. Konsentrasi dNTPs ditentukan oleh
panjang target DNA (100 µM).
2. MgCL2 (1,0-1,5 mM)
3. Polimerase DNA tergantung panjang
fragmen DNA yang akan di amplifikasi
(2 kb) 1,25-2 unit per 50 µL PCR reaksi.
 4) Thermostable DNA
Polymerase
1. DNA Polymerase is the enzyme responsible for
copying the sequence starting at the primer from
the single DNA strand.
2. Commonly use Taq, an enzyme from the
hyperthermophilic organisms Thermus
aquaticus, isolated first at a thermal spring.
3. This enzyme is heat-tolerant
– it is thermally tolerant (survives the melting T of DNA
denaturation)
– which also means the process is more specific, higher
temps result in less mismatch – more specific
replication
 Enzim/Tag Polimerase
1. Katalisis untuk reaksi polymerase
2. Ekstensi DNA
3. Terbuat dari bakteri termofilik (95°C)
 Buffer PCR
1. pH (low & high-salt buffer) 8,75
2. Target 0 -5 kb (Low-salt buffer)
3. Target > 5 kb (high-salt buffer)
 Running PCR
• The PCR is commonly carried out in a reaction volume
of 15-100 μl in small reaction tubes (0.2-0.5 ml
volumes) in a thermal cycler.
• The thermal cycler allows heating and cooling of the
reaction tubes to control the temperature required at
each reaction step.
• Thin-walled reaction tubes permit favorable thermal
conductivity to allow for rapid thermal equilibration.
• Most thermal cyclers have heated lids to prevent
condensation at the top of the reaction tube.
• Older thermocyclers lacking a heated lid require a
layer of oil on top of the reaction mixture or a ball of
wax inside the tube.
 Initialization step
• Prior to the first cycle, there is an
initialization step
– the PCR reaction is often heated to a
temperature of 94-96°C, and this
temperature is then held for 1-9 minutes
– This first hold is employed to ensure that
most of the DNA template and primers are
denatured,
– Also, some PCR polymerases require this
step for activation
 PCR Reaction Cycles
• One PCR cycle consists of a DNA denaturation
step, a primer annealing step and a primer
extension step.
 DNA Denaturation: Expose the DNA template to
high temperatures to separate the two DNA strands
and allow access by DNA polymerase and PCR
primers.
 Primer Annealing: Lower the temperature to allow
primers to anneal to their complementary sequence.
 Primer Extension: Adjust the temperature for
optimal thermostable DNA polymerase activity to
extend primers.
PCR
PCR: First 4 Cycles
PCR: Completed Amplification Cycle
PCR: Completed Amplification Cycle
 Each cycle: 1 copy of DNA template will
give 2 copies from double-stranded DNA
templates.
 n cycles will give 2n copies
 Assuming a cycle lasts 6 min:
 1 double-stranded DNA molecule
 35 cycles
 34x109 copies in 3.5 hrs!
 There are also ≈ 60 other DNA copies
 DNA copies vs Cycle number
2500000

2000000

1500000
DNA copies

1000000

500000

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Cycle number
 Analyze PCR products
• Check a sample by gel electrophoresis.
• Is the product the size that you
expected?
• Is there more than one band?
• Is any band the correct size?
• May need to optimize the reaction
conditions.
 PCR Modifications
• Nested PCR
– increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers
are being used in two successive PCRs.
• Multiplex PCR
– The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA
sequences.
• Reverse-transcriptase PCR
– (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The
PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA.
 Polymerase Chain Reaction
Controls for PCR
• Blank reaction (Negative control reaction)
– Controls for contamination
– Contains all reagents except DNA template

• Positive control reaction

– Controls for sensitivity
– Contains all reagents and a known target-containing
DNA template
 Procedure
1- Prepare master Mix
2- Program the thermocycler
3- Run the samples on thermocycler
4- Analysis of PCR products
KOMPONEN PCR
 Target DNA
• Amplification of part of the Human
growth hormone gene
• Specific primers used
• Forward primer: 5’-
TCCCTTCCCAACCATTCCCTTA-3’
• Reverse primer: 5’-
CCACTCACGGATTTCTGTTGTGTTTC-
3’
 1- Master Mix
PCR reaction mixture
Reagent Volume (µl) Final concentration
PCR buffer (X10) 2.0 10 mM
MgCl2 (25 mM) 1.6 2.0 mM
dNTPs (100mM) 0.1 0.1 mM
Primer 1 (F) 0.2 1.0 µM
Primer 2 (R) 0.2 1.0 µM
Taq DNA
0.25 2.0 U
polymerase
DNA template 2.0 100 ng
Water 13.7
Nested-PCR
2- Program the Thermocycler
The Thermocycler Profile is:
Step 1: Denaturation for 3 min. at 95oC
Step 2: 35 cycles
Melting for 60 sec. at 95oC
Annealing for 60 sec. at 57oC
Extension for 90 sec. at 72oC
Step 3: Final elongation for 10 min. at
72oC
 4- Analysis of PCR products

 Analyse products on 2% +ve Sample

-ve
agarose gel containing
ethidium bromide
 Visualize the PCR product
on UV transilluminator
Permasalahan Pada PCR
Could You Interpretation This PCR Result
 Contamination of PCR Reactions

1. Most common cause is carelessness and bad

technique.
2. Separate pre- and post-PCR facilities.
3. Dedicated pipettes and reagents.
4. Change gloves.
5. Aerosol barrier pipette tips.
6. 10% bleach, UV light
Target (Template)
Primer
Reaction Components
Thermal cycling conditions
Electrophoresis and staining
Primer-Dimer