PCR ELISA

Presented to
Dr. RIDA BATOOL
Presented by
Tehreem Ehsan (MSBT-05F21)
Fatima Iqbal (MSBT-0721)
Muhammada Jabeen (MSBT-08F21)
Tayyaba Nasir (MSBT-09F21)
Uswa Fatima(MSBT-14F21)
 CONTENTS

PCR ELISA

History

Need for PCR ELISA

Principle of PCR ELISA

Protocol of PCR ELISA

Comparison of PCR ELISA with other PCR technologies

Applications of PCR ELISA

 CONTENTS

Advantages of PCR ELISA

Disadvantages of PCR ELISA

Recent advances in PCR ELISA

Conclusion

References
 PCR ELISA

• “An immunological method to quantify the PCR product directly after immobilization
of biotinylated DNA on a microplate.”

• Also known as PCR-ELOSA (polymerase chain reaction-enzyme-linked oligosorbent

assay).

• This technique originated in the 1980’s and is most often used today in pathogen
detection.

• Used for detection of nucleic acids present in very low concentrations in biological
specimens.
MAIN STEPS

Amplification

Immobilization

Detection
HISTORY

In the early 90s, Friedrich Miescher first identified and isolated DNA.
In 1953 James D. Watson and Francis Crick discovered the double
helix structure of DNA .
In 1983 Kary Mullis invented polymerase chain reaction (PCR) , one
of the most innovative and still widely used techniques in the field of
life sciences.
HISTORY

In late 1980s, there was a sudden boom of interest in the study of
immune detection of DNA.
Immunodetection of DNA using enzyme linked immunosorbent assay
techniques were published, leading to the introduction of PCR-ELISA.
This method combines both PCR and ELISA into a single analytical
technique and its application is very much similar to ELISA except that
this method allows the detection of nucleic acid instead of protein.
1869  DNA was first identified by Swiss chemist Friedrich Miescher.

1953 James D. Watson and Francis Crick first discovered the double helix structure of DNA.

1968 The principle of replicating DNA by primers was discovered by Gobind Khorana.

1971 Eva Engvall and Peter Perlman (independently) invent a method that revolutionized medicine
called the ELISA test. 
1983 PCR was invented by the American chemist, Kary Mullis.

1980s There was a sudden boom of interest in the study of immunodetection of DNA.

1989 Coutlée et al. studied the immunodetection of DNA using biotinylated RNA probes.
NEED FOR PCR ELISA

• Conventional ELISA is used to detect antigens and circulating antibodies in serum.

• Despite of its versatility, ELISA is hampered by the requirement for purified

antibodies and antigens, which is time consuming to generate and require extensive
optimization.

• Instead, PCR-ELISA is capable of detecting genomic material from any biological

specimen, as long as suitable primers can be designed to amplify the target DNA
sequence.
PRINCIPLE

A sensitive and specific PCR–ELISA involves the labeling of

target amplicons during PCR, their hybridization with target-
specific probes, capture of labelled hybrids onto microtiter
plates or tubes and subsequent detection by immunoassay.
COMPARISONS OF PCR-ELISA WITH OTHER
PCR-BASED MOLECULAR APPROACHES
 Comparison Conventional PCR PCR-ELISA qPCR

Equipment required Standard laboratory equipment Standard laboratory equipment Requires fluorescence detection
instrument

Reagent costs Low Moderate Costly

Quantitative Not-quantitative Semi-quantitative Quantitative

Specificity Low High Moderate

       
 Detection limit      
1–10 ng/μL 0.01 ng/μL 0.25 pg/μL
 
 
Carcinogen material Methylene blue Not carcinogen Florescence material
 CHEMICALS AND APPARATUS

• Target sequence

• Consensus Primers

• Biotinylated Probe

• Dig dNTPs

• Thermal Cycler

• Streptavidinated Microtiter Plate

• Streptophotometer
 Detection Primer
design

Immobilization Probe
STEPS design

Amplification
Hybridization
 PROTOCOL

1. PRIMER DESIGN
• Primers are short pieces of nucleotides used in the initiation of DNA synthesis.
• Consensus primers are required to span a conserved genomic region within the
bacterial genus/genera with enough variability to discriminate between species.

2. PROBE DESIGN

Probes are generated using biotinylated primers.

3. PCR AMPLIFIES TARGET SEQUENCE
Target sequences are amplified using thermocycler. Amplicons are labeled with
digoxigenin (DIG) molecules so that they can later be detected with highly specific
anti-DIG antibodies.

4.HYBRIDIZATION
PCR amplicons from test samples are incubated with biotinylated probes under
conditions that allow sequence-specific hybridization of DNA molecules.
5. COATING WITH STREPTAVIDIN
The plates are then coated with streptavidin which has a very strong affinity for
biotin and therefore binds to the biotinylated probes.

6. IMMOBILIZATION
The hybrids are captured on the microtiter plate via streptavidin-biotin binding.
The biotinylation of probes enhances specificity by ensuring that only amplicons
bound to their specific probes are immobilized on this plate.
7. Addition of anti-DIG antibody
Anti-DIG antibody recognizes DIG-hybrids and remains on the plate. The
antibody is usually conjugated to peroxidase or alkaline phosphatase.

8. Addition of colorimetric substrate

The substrate will undergo a color change in the presence of conjugate, and this
can be measured spectrophotometrically. Between each step, the microtiter plate is
washed with a mild detergent solution to remove any unspecific hybrids or
antibodies.
Figure 2. Schematic diagram of the PCR DIG-ELISA on a microtiter plate.
Figure 3. Scheme of PCR–ELISA. DIG-labelled target sequences (amplicons) are hybridized with specific
biotinylated probes. Biotinylated DIG-labeled hybrids are immobilized onto a microtiter plate via biotin-streptavidin
binding. Addition of specific anti-DIG antibody conjugate produces a colour change in the substrate, which is
quantified spectrophotmetically.
 ADVANTAGES OF PCR ELISA

Semi-quantitative
Specificity
Technique

Lack of
High Sensitivity Carcinogenicity

Detection in Short
Cost Effective
Time
SEMI-QUANTITATIVE TECHNIQUE

Appearance of colour
Colour Intensity

High SENSITIVITY

Highly sensitive as compared to conventional PCR

Sensitive to extent of nano or picogram per µl
Specificity

Specific gene design PCR-ELISA

 Unique DNA sequence of each organism
 Use of specific primers and probe sequence

DETECTION IN SHORT TIME

 Less time consuming

 Procedure time: 4 hours
 Easier to perform
LACK OF CARCINOGENICITY

 No carcinogenic material in PCR-ELISA

Automatable

Robust standardization of methods, reagents and samples

SIMULTANEOUS DETECTION OF A
LARGE NO. OF SAMPLES

 Depends on the numbers of wells in PCR-ELISA kit

 No need of high expertise

COST EFFECTIVE

 Use of standard laboratory equipment

 Rule out the need for expensive real-time PCR machines
and DNA sequencing
APPLICATIONS OF PCR ELISA
 DETECTION AND DIAGONESIS

 Detection at low concentration

 Detection of latent symptoms of diseases

 Gene expression studies through biomarkers

 Employed for cancer cells, viruses, etc.

 QUANTITATIVE MONITORING

 Detection of a particular substance

 Quick indication assay
 Monitoring of:
 Cytomegalovirus infection in bone marrow transplant
recipients
 Fibrobronchoscopic biopsy in lung cancer patients
 Quantitative monitoring of Leishmania parasite in livestock
 Detection and
Diagnosis
logy
io ive
m t
pi de
n t ita g
E a in
Qu nitor
Geno Mo
ty
of ind ping
ividua
ls Env
ir
Mon onment
itori al
Di ng
of a gn
vir osi
us s Blood
screening
 Requirement of
Laborious Technique
Primers and Probes

DISADVANTAGES It is a laborious
PCR-ELISA cannot be technique as it
involves various steps
used to detect
such as amplification,
unknown targets. immobilization, and
detection.
  Mutations in PCR products

 Optimization of Primer pair

LIMITATIONS
OF PCR ELISA  Redundancy in Immunological research

 Redundancy in the diagnosis of immune

disorders and Cancer

R P C R E L I S A
E
C  Due to its ability to test multiple samples in a single run with a shorter running

E time, a number of PCR-ELISA trial runs are currently in progress for use in
N
T medical diagnosis.
A  With the success of these trial runs, it can be foreseen that the assay would be used
D
V in various quality control and medical diagnostic labs in the near future.
A  Development of multiplexing technique.
N
C
ES T E C H N O L O G Y
C
O
N
C
L
U  PCR ELISA is a immunodetection method that detects
S nucleic acids instead of proteins with vast applications in
medical, veterinary and agricultural industries.
I  The high sensitivity and specificity of PCR-ELISA make it a
powerful technique by simple laboratory facilities
O
N
REFERENCES

 Al Dahouk, S., Tomaso, H., Nöckler, K., & Neubauer, H. (2004). The detection of Brucella
spp. using PCR-ELISA and real-time PCR assays. Clinical laboratory, 50(7-8), 387-394.
 Coutlée, F., Bobo, L., Mayur, K., Yolken, R. H., & Viscidi, R. P. (1989). Immunodetection
of DNA with biotinylated RNA probes: a study of reactivity of a monoclonal antibody to
DNA-RNA hybrids. Analytical biochemistry, 181(1), 96-105.
 Hajia, M. (2018). Limitations of different PCR protocols used in diagnostic laboratories: a
short review. Modern Medical Laboratory Journal, 1(1), 1-6.
 Joshi, M., & Deshpande, J. D. (2010). Polymerase chain reaction: methods, principles and
application. International Journal of Biomedical Research, 2(1), 81-97.
 Menotti, J., Garin, Y. J., Thulliez, P., Sérugue, M. C., Stanislawiak, J., Ribaud, P., ... &
Derouin, F. (2010). Evaluation of a new 5'-nuclease real-time PCR assay targeting the
Toxoplasma gondii AF146527 genomic repeat. Clinical Microbiology and Infection, 16(4),
363-368.
REFERENCES

 Sue, M. J., Yeap, S. K., Omar, A. R., & Tan, S. W. (2014). Application of PCR-ELISA in molecular
diagnosis. BioMed research international, 2014, 653014. https://doi.org/10.1155/2014/653014
 Tayebeh, F., Nazarian, S., Mirhosseini, S. A., & Amani, J. (2017). Novel PCR-ELISA technique as
a good substitute in molecular assay. Journal of Applied Biotechnology Reports, 4(2), 567-572.
 Musiani, M., Venturoli, S., Gallinella, G., & Zerbini, M. (2007). Qualitative PCR–ELISA protocol
for the detection and typing of viral genomes. Nature Protocols, 2(10), 2502-2510
 Polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) retrieved on
October 26, 2014, from
https://bitesizebio.com/20445/polymerase-chain-reaction-enzyme-linked-immunosorbent-assay-pc
r-elisa
/
 Enzyme-linked Immunosorbent Assays (ELISA): Recent Innovations Take Analyte Detection to
New Levels by Dr. John Comley retrieved on October 6, 2012, from
https://www.ddw-online.com/enzyme-linked-immunosorbent-assays-elisa-recent-innovations-take-
analyte-detection-to-new-levels-1690-201210
/