Application of PCR on Infectious Diseases

Application of PCR in
Detecting Infectious
Diseases
Department of Pharmaceutical Sciences
North south University, Bangladesh.

Group Members
ID: 1620793049
Indrajit Barua Muthsuddy
01
ID: 1621744049
Md. Ismayel hossain
02
ID: 1620412049
Labony Sarker
03
ID: 1621297049
Sharmine Akter
04
ID: 1621776049
Md.Abu Anam
05

 It is the most widely used target nucleic
acid amplification method.
 PCR is an enzymatic process in which a
specific region of DNA is replicated over
and over again to yield many copies of a
particular sequence.
Polymerase Chain
Reaction (PCR)

01
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• DNA Polymerase: 1-2.5 units
• Primers: 0.1-0.5 µM
• dNTPs: 20-200 µM
• Target DNA: 1 µg
• Buffer: pH 8.3-8.8
• Magnesium chloride: .5-
2.5mM
A Standard PCR Reaction Mix

It is the most well-
developed molecular
technique up to now.
PCR has revolutionized the
clinical practice of infectious
disease.
It has a wide range potential, clinical applications, including specific
or broad-spectrum pathogen detection, evaluation of emerging novel
infections, surveillance, early detection of biothreat agents.
PCR
For infectious disease critical
and timely intervention relies
on rapid and accurate detection
of the pathogen in the acute-
care setting.
PCR played an important role in the recent
outbreak like COVID-19 (SARS-CoV-2), SARS,
MERS, ZICA Virus, Anthrax, HIV etc.
Either DNA or RNA (following the
production of complementary DNA using
reverse transcriptase) can be used as a
template for amplification.
PCR and Infectious Disease

Diagnostic Goals for PCR
PCR has been used for three broad categories of diagnostic
goals -.
Detection
(Is the target nucleic acid present in the specimen?)
Characterization
(Which variant of the target nucleic acid is present?)
Quantitation
(How many copies of the target nucleic acid are
present?)

Infectious Diseases Diagnosed by PCR -
 Hepatitis C Virus
 Hepatitis B Virus
 Human Papilloma Virus
 Zika Virus
 Meninggitis
 Encephalities
 Myelitis
 COVID 19
 SARS
 MERS
 Anthrax
 HIV, Types 1 and 2
 HTL V, Types I and II
 Tuberculosis

Advantages of PCR Over Conventional Diagnostic
Methods -
High Sensitivity - Under the right conditions, one can amplify a single copy of a microbial
DNA gene or gene fragment from a clinical sample and detect it.
High Specificity - PCR offers the potential for remarkable specificity.
High Speed - PCR can be completed in minutes to hours.
Robustness

Different Types of PCR
There are Different types of PCR .
 Real-Time PCR -
Quantitative PCR (qPCR)
 Reverse Transcriptase
PCR (RT-PCR)
 Reverse-Transcriptase
Real-Time PCR (RT-qPCR)
PCR used in Diagnosis of
different Diseases-
 Alu PCR
 Assembly PCR
 Asymmetric PCR
 COLD PCR
 Colony PCR
 Nested PCR
For Example –
Clinically Used
Commonly Used

Reverse Transcriptase PCR (RT-PCR)
Reverse transcription polymerase
chain reaction (RT-PCR) is a
laboratory technique combining
reverse transcription of RNA into
DNA and amplification of specific
DNA targets using polymerase
chain reaction (PCR).

Reverse Transcriptase PCR (RT-PCR)
Step 1
Step 2
Step 3 Step 5
RNA is extracted from
tissue
Analyze results01 03 05
02 04
Step 4
RNA template is converted
into a complementary DNA
(cDNA) using reverse
transcriptase.
The cDNA then acts as a
template for exponential
amplification using PCR.
RT-PCR can be conducted either
in a single tube or as two steps in
different tubes.
.

Limitations of PCR
03
04
05
02
01
Highly qualified technicians are
needed
Sample Acquisition and Preparation
False Negatives
False Positives
High Cost

ID: 1621744049
Md. Ismayel hossain

CORONA VIRUSES:
• An acute respiratory disease, caused by a novel coronavirus SARS-COV-2, previously
known as 2019- nCOV, the COVID-19 has spread throughout china and received worldwide
attention.
• On 30 January 2020, WHO officially declared the COVID-19 Epidemic as a public health
emergency of international concern.

CORONA VIRUSES..
Corona viruses are single-stranded
RNA viruses, about 120 nanometers
in diameters.
They are susceptible to mutation
and recombination, therefore they
are diverse.
There are about 40 different
varieties and they mainly infect
human and non-human.
The corona-like appearance of
coronaviruses is caused by spike
glycoproteins which are necessary
for the viruses to enter host cells.

CORONA VIRUSES..
The Covid-19 affects
different people in
different ways.
Most infected people
will develop mild to
moderate illness and
recover without
hospitalization.
Most common
symptoms includes-
fever, dry cough,
tiredness. Serious
symptoms difficulty
breathing or shortness
of breath.
So far, worldwide
scenario: Total cases
38.6 M, Recovered
26.7 M and deaths
1.09M.

ROLE OF POLYMERASE CHAIN REACTION ON
COVID-19 TEST:
• Thus, far the most commonly used and reliable test for diagnosis of covid-19
has been the RT-PCR test because they are very robust tests generally, with
a low false-positive and a low-false negative rate.
Feature Value
Sensitivity ( The true
positive rate)
89%
Specificity 91%
Positive predictive
vale ( PPV)
97%
Negative predictive
value ( NPV)
72%
False detection rate 3%
False omission rate 28%

POLYMERASE CHAIN REACTION..
In this test, the virus’s single-stranded
RNA is converted to its complementary
DNA by reverse transcriptase; specific
regions of the DNA, marked by primers,
are then amplified.
This is done by synthesizing new DNA
strand from deoxynucleoside
triphosphate using DNA polymerase.
The Real time RT-PCR Diagnostic panel
is a molecular in vitro diagnostic that
aids in the detection and diagnosis
2019-nCOV and is based on widely
used nucleic acid amplification
technology.
Testing with the 2019-nCOV Real-Time
RT-PCR Diagnostic panel is intended for
use by trained laboratory personnel who
are proficient in performing real-time RT-
PCR assays.
RT-PCR

MATERIAL INCLUDED IN RT-PCR DIAGNOSTIC PANEL:
Three primer-probe mixes for:
1. 2019-nCov_N1
2. 2019-ncov_N2
3. RNase P gene
2019-ncov_N1 and 2019-ncov_N2:
Target virus nucleocapsid (N) gene for
specific detection of SARS-COV-2
RP: targets human Rnase P gene for
detection of human nucleic acids; control
for sample integrity.
nCovPC; Non-infectious positive control
material
Human Specimen Control ( HSC): used
as an extraction procedural control.
No Template control (NTC): Nuclease-
free water included in each run.

RT-PCR PROCESS:
1. A primer is attached to the 3’ end of a
single strand of viral RNA.
2. Deoxynucleoside triphosphates are
added stepwise
3. Creating a DNA copy of the viral RNA.
4. The single strand of DNA is separated.
5. Double stranded complementary DNA is
prepared.
6. Copies of which are synthesized using
primers and DNA polymerase.

PRINCIPLES OF THE PROCEDURE:
The oligonucleotide primers and probes for detection of
2019-nCOV were selected from regions of the virus
nucleocapsid (N) gene. The panel is designed for specific
detection of the 2019-nCOV. Initially two primers sets is
used. An additional primer set to detect the human Rnase
P gene in control samples and clinical specimens is also
included in the panel
Then, RNA isolated and purified from upper and lower
respiratory specimens is reverse transcribed to cDNA and
subsequently amplified in the applied bio systems 7500
Fast DX Real-Time PCR instrument with SDS version 1.4
software.
In the process, the probe anneals to a specific targets
sequence located between the forward and reverse
primers. During the extension phase of PCR cycle, the
5’ nuclease activity of Taq polymerase degrades the
probe, causing the reporter dye to separate from the
quencher dye, generating a fluorescent signal.
With each cycle, additional reporter dye molecules are
cleaved from their respective probes, increasing the
fluorescence intensity. Fluorescence intensity is
monitored at each PCR cycle by Applied Biosysytems
7500 Fast DX Real-Time PCR system with SDS version
1.4 software. Detection of viral RNA not only aids in the
diagnosis of illness but also provides epidemiology and
surveillance information.
RT-PCR

SUMMARY OF PREPARATION AND TESTING PROCESS:
Resuspend primer/probe mix, aliquot and store at < - 20
degree
Resuspend and aliquot nCOVpc, store at -70 degree. ( Upon
receipt of rRT-PCR panel reagents)
Upon obtaining sample- Extract sample RNA and HSC RNA
Prepare master mix ( 15 micro liter) and prepare rRT- PCR
plate ( 5 micro liter RNA)
Then, Run assay on ABI 7500 Fast DX
Then, Data analyze and report the results.

2019-NCOV MARKERS:
A specimen is considered negative if all
2019-nCOV marker ( N1, N2) threshold
growth curves do not cross the
threshold line within 40 cycles and the
Rnase P growth curve does cross the
threshold line within 40 cycles.
A specimen is considered positive for
2019- nCOV if all 2019-nCOV marker (
N1,N2) cycle threshold growth curves
cross the threshold line within 40 cycles
( < 40.00 ct)
When all controls exhibit the expected
performance and the growth curves for
the 2019-nCOV markers (N1,N2) and
the Rnase P marker do not cross the
cycle threshold growth curve within 40
cycles ( < 40 ct), the result is invalid.
When all controls exhibit the expected
performance and the growth curves for
the 2019-nCOV markers and the Rnase
cross the cycle threshold growth curve
within 40 cycles, the result is
inconclusive.
RT-PCR

2019-NCOV RT-PCR DIAGNOSTIC PANEL RESULTS:
2019
nCOV_N1
2019
nCOV_N2
RP Result
Interpretatio
n
Report
+ + +,- 2019-nCOV
detected
Positive 2019-
nCOV
If only one of
the two
targets is
positive
If only one of
the two target
is positive
+,- Inconclusive
Result
Inconclusive
- - + 2019-nCOV
Not Detected
Not Detected

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 HIV is a virus that damages the cell in
immune system that potentially leads
to life threatening condition that
weakens ability to fight infections and
diseases.
 The two human immunodeficiency
viruses, HIV-1 and HIV-2, are members
of the family of Retroviruses, in the
genus of Lentiviruses.
HIV virus
detection by PCR

The methods of diagnosing HIV
Screening test(antibody
detection)
o ELISA test
o Rapid sample test
Supplemental test (antibody
detection)
o Western blot assay
o Immunofluuroscence assay
Confirmatory test
o P24 antibody detection
o HIV RNA PCR (the best confirmatory method)
 Reverse transcripted PCR
 Branched DNA assay
 Neucleic acid base amplification
 Real time RT-PCR for estimating viral load

HIV-PCR test can detect virus
earliest; within 7-11 days
When screening test and supplementary
tests are failed or can not be relied.
When PCR is conducted to detect HIV?

Collecting the
blood sample
Extracting
the mRNA
required
From the isolated mRNA
and collecting the specific
gene is collected that
responsible for viral HIV
antigen
Amplifyin
g the gene.
The process of conducting HIV detection by PCR

 Time saving, less labour needed
 Provide detection of actively replicating cells
also transcriptionally dormant cells.
 It is called the best confirmatory method; gold
standard method
Advantage of HIV virus detection by PCR
Clinical specificity
>99%
Clinical sensitivity
N/A

• Infected individuals prior to the generation of antibodies
• Resolving the infection status of individuals with ambiguous or
indeterminate serological status using the Western blot assay
• Documenting infection in seropositive individuals negative by other direct
detection assays
• Determining the type of virus present.
• Differentiate latent HIV from active
• Useful for diagnosis of pediatric
• Provirus detect
• Detect window period, virus load estimation, detect geno types
Clinical Utility of Identifying HIV Proviral Sequence by PCR

Human T cell lymphotropic virus (HTLV)
• Human T cell lymphotropic virus (HTLV)
infects a type of white blood cell called a T-
cell or T-lymphocyte. HTL V-1 is a member
of the Oncovirinae subfamily of retroviruses
• It causes diseases like T cell leukemia and
tropical spastic paraparesis (tSP).

• The presence of HTLV can be detected by a blood test.
• There are three tests for HTLV:
1) ELISA
2) Confirmatory Blot
3) PCR
• WHEN PCR test is conducted test looks directly for HTLV proviral
DNA in the blood cells. This reports the percentage of peripheral
blood mononuclear cells (PBMCs) which carry the virus.
For example if the PCR test says
the HTLV proviral load is 1%, then
this means that 1 out of 100
PBMCs have one copy of HTLV
DNA incorporated into the DNA.

• Identifying infected individuals prior to seroconversion
• Confirming infection in seropositive individuals
• Documenting infection in individuals presenting with symptoms
dissimilar from classic ATL
• Typing the virus present (ex- type I versus type II).
Clinical Utility of Identifying HTLV Proviral Sequence by PCR

Hepatitis B
Hepatitis B is an infectious disease caused by the
hepatitis B virus (HBV) that affects the liver; it
is a type of viral hepatitis.

PCR based diagnosis of HBV
The detection of
viral genome
through the use of
PCR has made
possible a new
step in viral
diagnosis.
The main
advantages of PCR
are its extreme
sensitivity and the
possibility to
develop rapid
assays using non
radioactive probes.
The amplification
of hepatitis B
virus DNA
sequences in sera
for the diagnosis
of HBV is an
important
application of
PCR with regard
to HBV.

Process of PCR based diagnosis of HBV
This procedure will be more reliable evidence of the presence of Hepatitis B virus.
A set of primer B2833S
GGGTCACCATATTCT
TGG and B170AS GT
CCTAGGAATCCTGAT
G was tested for the
amplification of HBV
genome from the plasma
derived HBV patients and
a cloned HBV.
Annealing
temperature and
units of Taq
polymerase were
optimized.
Annealing
temperature 55°C
was found best for
B2833S and
B170AS.
It was established
that one unit of Taq
polymerase amplify
optimally in a
reaction mixture of
25 µl.

Limitations of HBV PCR
False positives by contamination and false negatives by quality of DNA and RNA
Defective genomes
Contamination of tissue samples by serum particles
Specificity
>98%
Sensitivity
N/A

Tuberculosis
• Tuberculosis is an infectious disease usually caused by
Mycobacterium tuberculosis bacteria.
• Tuberculosis generally affects the lungs, but can also
affect other parts of the body.

TB-PCR
The TB-PCR is one
of the tests that helps
diagnose and
confirm an infection
of Tuberculosis
The TB-PCR test is
ordered to diagnose
and detects
M.Tuberculosis from
other mycobacteria
It also helps
monitor treatment
for TB.
PCR test based on
insertion sequence
IS1081 was developed to
detect Mycobacterium
tuberculosis complex
organisms in the
peripheral blood.
The method was
applied to blood
samples from
immunocompetent
individuals with
localized pulmonary
tuberculosis

Process of TB-PCR
First of all, Five milliliters of sample was collected in EDTA-anticoagulated tube.
Leukocytes were pelleted after the lysis of erythrocytes by a hypotonic erythrocyte
lysis buffer under chilled conditions. These leukocytes were resuspended in 1 ml of
Tris-EDTA buffer.
The leukocyte samples were subjected to supercooling in a liquid nitrogen container
for about 15 min and then thawed abruptly in a boiling water bath for 10 min.
DNA was precipitated with 0.6 volume of isopropanol in the aqueous phase and
separated by chloroform treatment and extraction.

Process of TB-PCR
Then, primers aimed at the 306-bp region of the multicopy insertion sequence IS1081
were used to amplify mycobacterial DNA in the blood leukocyte samples.
The amplification parameters included an initial denaturation at 94°C for 5 min
followed by 35 cycles each of denaturation at 94°C for 1 min, annealing at 68°C for
1.5 min, and extension at 72°C for 2 min.
The extension step in the 35th cycle was held for 10 min before the samples were
shifted to 4°C for storage.

Sensitivity and Specificity
79.4-91.9
Sensitivity
>99
Specificity

Limitation of TB-PCR
Chance of false-positive result is higher.
Chance of infection is higher.

Cytomegalovirus Infections
Pathogenic Microbe:
Cytomegalovirus (CMV)
This viral infection can lead to mental retardation and nonhereditary
sensorineural deafness.
Early detection of this virus is important for chemotherapy.
PCR can detect CMV in urine specimens of infected with greater
sensitivity than virus culture.
Technique : Real-time PCR.
Machine : Light cycler PCR machine.

Light Cycler
 Sensitivity : 9719
 Limitation:
 Temperature cycle can’t be maintained.
 Results are not given for the samples which
cannot give fluorescence during detection.
It enables the user-
 to monitor the amplification of the PCR
product
 in real-time allows the accurate quantification
of target sequences.

Process
First identify two sets of primers, one
specific for CMV and the other specific
for human DNA
the CMV primers were directed at a
conserved region of the DNA polymerase
gene of strain
The forward primer was 5′-GCT GAC GCG TTT GGT CAT C
and the reverse primer was 5′-ACG ATT CAC GGA GCA CCA
G.
The internal probe was labeled at the 5′ end with the
fluorescent dye 6-carboxyfluorescein and on the 3′ end with
the quencher dye 6-carboxytetramethylrhodamine (TETRA)
Then in light cycler it gives specific
fluorescence which helps to detect the target
sequence of CMV.

Human papillomavirus infection
Pathogenic Microbe:
Human papillomaviruses (HPVs).
 This virus is associated with a number of diseases and cancers such
as-
• cervical dysplasia and carcinoma
• benign condylomas
Technique : Multiplex PCR with Type-Specific Primers can be
used.
Machine : Thermal cycler.

Thermal cycler
 It is used to amplify the segments of
DNA via PCR .
 It is programmed to carry out heating
and cooling of samples over a number
of cycles.
 Sensitivity: 9819
 Limitation:
 Only used to identify the presence or
absence of a known pathogen or gene

Process
1. Those women who had cervical lesion , the samples were taken using of sterile cotton swab,
and were stored at -20ºC and compared five different extraction protocols(A to D)
2. Protocol A is based on freezing and boiling of the samples and
3. In protocol B, a commercial kit for DNA extraction based on precipitation of proteins where the
solution is mixed by pipetting and incubated at 37°C for 1 h
4. Protocol C is based on proteinase K digestion of the samples which is incubated at 56°C for 2 h
5. Protocol D includes organic extraction incubation at 37°C for 1 h.

Future Application
In the identification of human retroviruses and their association with
disease, particularly is in its infancy.
Since related viruses share short hyphenated regions of homology, can be
used to facilitate the detection of uncharacterized viruses.
In the development of procedures to convert PCR to a quantitative assay
To assist in the evaluation of therapeutic trials for antiviral intervention.
In model studies, coincident detection of members of Oncoviruses and
hepadnaviruses.

Reference
1. Erlich HA. PCR technology. Principles and applications for DNA amplification. 1989:61-
70.
2. Yang S, Rothman RE. PCR-based diagnostics for infectious diseases: uses, limitations,
and future applications in acute-care settings. The Lancet infectious diseases. 2004 Jun
1;4(6):337-48.
3. Yang S, Rothman RE. PCR-based diagnostics for infectious diseases: uses, limitations,
and future applications in acute-care settings. The Lancet infectious diseases. 2004 Jun
1;4(6):337-48.
4. Fredricks DN, Relman DA. Application of polymerase chain reaction to the diagnosis of
infectious diseases. Clinical infectious diseases. 1999 Sep 1:475-86.
5. Frothingham R. Applications of the polymerase chain reaction to infectious disease
diagnosis. Annals of Saudi medicine. 1996 Nov;16(6):657-65.

Application of PCR on Infectious Diseases

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