The Principle of the RT-PCR test
B.Undarmaa
OUTLINE
I.
Brief introduction to Molecular Biology section
II. Molecular Biology equipment
III. Basics of Molecular Biology
IV. What is PCR ?
V. Briefly about nucleic acid extraction
VI. RT-PCR in brief
VII.Cautions
1. Brief introduction to Molecular
Biology section
I. Brief introduction to Molecular Biology section
• RT-PCR
• STD 7 pathogens
• Vaginitis screening assay- 7
• Clinical pathologist-1
• Biomedical scientists-4
• Laboratory technicians- 3
In total 3 methods are used in 14
different lab orders.
•
•
•
RT-PCR
SARS-CoV-2 assay
Respiratory 8 types of viruses
• Rt-PCR
• Monkeypox assay
• H.pylori/Clari/R assay
• Nested-PCR /Filmarray/-onestep
• Respiratory panel with/ without SARS-CoV-2
• Meningitis/Encephalitis panel
• Rt-PCR melting point analysis-onestep/ GeneXpert
• HBV DNA, HCV-RNA, MTB/RIF assay
• Rt-PCR melting point analysis
• HPV-14 High risk types
2. Molecular Biology equipment
HBV HCV MTB: 45-105 minutes
Molecular Laboratory section
The old equipment:
3. Basics of Molecular Biology
Basics of Molecular Biology
Genetic information contained in DNA
 DNA is built in a double helix
 DNA encodes the genetic
information (different genes,
different information)
 DNA is organized into a long chain
called chromosomes
 23 pairs of chromosomes are in the
nucleus of human cells
© Cepheid
Basics of Molecular Biology
DNA building blocks
DNA is made up of 4 nucleotides
– A = Adenine
– T = Thymine
G
A
T
– C = Cytosine
– G = Guanine
The 4 bases are linked together to form a sequence (single strand of DNA)
G C T T A T G C A T
7
© Cepheid
C
Basics of Molecular Biology
Basic Molecular Biology
Most DNA is double stranded and pairs in a unique way:
G C
Sugar Phosphate
3’
Linkages give a DNA
strand a Direction
Strands of a duplex
are anti parallel
8
© Cepheid
5’
T A
5’
C G A A T A C G T A
G C T T A T G C A T
3’
4.What is PCR ?
What is PCR ?
1. The tool usedPCR
(Polymerase Chain
Reaction) is a chain
(cyclic) reaction
which generates
copies of a specific
sequence of DNA
present in the
sample.
2. To replicate DNA is
the enzyme
polymerase. RNA can
also be a starting
product. /cDNA/
3. Primer-short set of
DNA that is specific
to certain gene
© Cepheid
RdRp/S gene
The extraction was manual:
5. Briefly about nucleic acid extraction
What is happening with the Real-Time PCR instrument?
Real-time PCR
instruments
consist of THREE
main components:
1. Thermal Cycler
(PCR machine)
2. Optical Module (to
detect
fluorescence in the
tubes during the
run)
3. Computer (to
translate the
fluorescence data
into meaningful
results)
• Manufacturer: Korea
Introduction
• Extraction
limit/time 32- 23 minutes
• Compatible automated system:
SeePrep 32
• Quality certificates
• Principle: Magnetic beads
Intended use:
•
•
•
•
•
•
•
•
Naso-pharyngeal swab
Oro-pharyngeal swab
Urine
Feces
Rectal swab
Sputum
Uro-genital swab/vaginal, cervical, urethral/
Liquid based cytology
• Lysis buffer- to lyse the
cell/detergent and
proteinase K/
• Binding buffer- Will be
added to the lysate a
suitable condition will
form for the nucleic acids
to bind with the
magnetic beads.
• WB1, WB2- /isopropanol
and ethanol to remove
cell debris/
• WB3 – Water based! To
remove the residual
ethanol from previous
step /also contains
magnetic beads/
• Elution buffer- DNA is
eluted in a low-salt
buffer to allow for pH
stabilization of the DNA
in storage. /DNA is water
soluble/
6. RT-PCR in brief
What is Real-Time PCR?
The Polymerase Chain Reaction (PCR) is a process for the
amplification of specific fragments of DNA.
Real-Time PCR a specialized technique that allows a PCR reaction to
be visualized “in real time” as the reaction progresses.
As we will see, Real-Time PCR allows us to measure minute amounts
of DNA sequences in a sample!
What is happening with the Real-Time PCR instrument?
Real-time PCR
instruments
consist of THREE
main components:
1. Thermal Cycler
(PCR machine)
2. Optical Module (to
detect
fluorescence in the
tubes during the
run)
3. Computer (to
translate the
fluorescence data
into meaningful
results)
What is Real-Time PCR used for?
Real-Time PCR has become a cornerstone of
molecular biology:
• Gene expression analysis
– Cancer research
– Drug research
• Disease diagnosis and management
– Viral quantification
• Food testing
– Percent GMO food
• Animal and plant breeding
– Gene copy number
• Forensic science
- Paternal determination
Master mix components
Components of a PCR reaction
GCTTATGCAT
• DNA template (virus, bacterial or human genome)
• Primers (oligonucleotides of about 21
nucleotides, which will anneal to the target DNA)
• Polymerase (a natural enzyme that can replicate DNA
once primers have annealed, eg: Taq polymerase)
• dNTP’s (a mix of all four nucleotides required to build
DNA: A, T, C, G)
• Buffer (Mg2+, Tris-HCl, Triton: providing the
optimal conditions for the polymerase to work)
• Probe (fluorophore and quencher: if seperates
from eachother, the fluorophore will emit light)
© Cepheid
T
G
A
C
Taq polymerase
RT-PCR
Taqman Probe
GCTTATGCAT
T
G
A
C
Taq polymerase
PCR steps
DNA
or
RNA
PCR STEPS: first phase of a PCR cycle - Denaturation
denaturation
separation of DNA strands
• 90-95 ºC
• 20-30 sec.
PCR STEPS: second phase of a PCR cycle Annealing
annealing
specific primer binding
Temperature: 40-65 ºC
Duration: 20-40 seconds
5
© Cepheid
PCR STEPS: 3rd phase of PCR cycle- extension
GCTTATGCAT
extension
5’. Reverse primer
3’
Pol
3’
5’ Forward primer
• 60-75 ºC
Duration depends
on the sequence size
to be amplified
© Cepheid
Баднаа
5000000
4500000
4 units
Ct=23
Доржоо
4000000
3500000
3000000
2500000
2000000
1 unit
Ct=25
Аюуш
1500000
1000000
500000
0
0
1/8 unit
Ct=28
5
10
15
20
25
30
35
40
Let’s imagine that you start with four times as
much DNA as I do…picture our two tubes
at cycle 25 and work backwards a few
cycles.
Imagining Real-Time PCR
Cycle 25
Measuring
Quantities
Cycle
Me
You
23
250,000
1,000,000
24
500,000
2,000,000
25
1,000,000
4,000,000
LOD: H.pylori- >50 copies/reaction
ClariR- >100 copies reaction
• Respiratory 8 types of viruses
• STD-7
• Vaginitis screening-7
• HPV-14
Polymerase Chain Reaction Evolution
• Monkeypox
Homebrew PCR
• COVID-19 PCR
Batch
Xpert Tests
Patient Sample(s)
Patient Sample(s)
Washing
Sample Preparation
Nucleic acid extraction
Purification
• HCV/HBV/MTB/RIF NAAT
MasterMix
Amplification and Detection
Result Interpretation by Lab Technician
3
4
• Respiratory panel 17
• Respiratory panel with SARS-CoV-2
• Meningitis/Encephalitis panel-14
Automatic Result Interpretation
Flexi
Batch
7. Cautions
Cautions ⚠
• Working area and the benchtop should be
clean
• Work with clean gloves
• Pre-PCR and Post PCR items should not be
mixed
• All plastic wears should be RNAse and DNAse
free
THANK YOU SO MUCH