PCR

1. POLYMERASE CHAIN REACTION (PCR)
GOOD, BAD OR UGLY ?

2. Requirements for an effective diagnosis of
Infectious diseases
 Diagnostic tests that are rapid, reliable and highly
sensitive & specific
 Direct cultivation of pathogen
 Some pathogens are not cultivable
 Cell lines suitable for virus culture
 Development of immunoassay and DNA-based
diagnostic methods

3. Concept of DNA based methods
 Each species of pathogen carries unique DNA or RNA
sequences that differentiates from other pathogens
 These methods are highly sensitive and specific
 Allows rapid detection of pathogens
 Finds increasing use in diagnostic microbiology Lab

4. Nucleic acid amplification methods
 Polymerase chain reaction (PCR)
 Ligase chain reaction (LCR)
 Nucleic acid sequence based amplification (NASBA)
 Strand displacement amplification (SDA)
 Loop mediated isothermal amplification (LAMP)

5. Polymerase Chain Reaction (PCR)
Thermally controlled, enzyme mediated, invitro
amplification of the target DNA, with the help of
Primers, deoxyribonucleotide triphosphates
(dNTPs) and buffers at optimal concentrations

6. Dr. Kary Mullis, wins Nobel Prize in 1993
 Nobel Prize in chemistry
for his discovery of PCR
 The process was
conceptualized in 1983
 Considered as one of the
milestones among
scientific techniques of
the twentieth century

7. STEPS IN PCR
 EXTRACTION OF DNA
 AMPLIFICATION OF TARGET DNA
 DETECTION OF THE AMPLICONS

8. DNA EXTRACTION
METHODS
• Phenol and chloroform
• Cetyl Trimethyl Ammonium Bromide (CTAB)
• Column
• Guanidine isothiocyanate
• Sorbent

9. AMPLIFICATION
 MASTER MIX
• Primers
• Buffers
• Taq DNA polymerase
• dNTPs
• Water (PCR grade)
 THERMO CYCLER

10. Concentration of ingredients in PCR
master mix
 Magnesium chloride: 0.5-2.5 mM
 Buffer: pH 8.3-8.8
 dNTPs: 20-200 µM
 Primers: 0.1-0.5 µM
 DNA Polymerase: 1-2.5 units
 Target DNA:  1 µg

11. STEPS IN AMPLIFICATION
 Denaturation
930 - 950C for 30 - 60 sec
 Primer annealing
550 - 600C for 20 - 60 sec
 Primer extension / elongation
700 - 75°C for 30 - 60 sec

12. PCR animation

13. Post Amplification Detection Methods
 By agarose gel electrophoresis
 Enzymatic detection of amplified product
(capture probe)
 Reverse hybridization (oligonucleotide probe)
 Sequencing of the amplicons

14. Agarose gel electrophoresis
animation

15. M Nce Pc 1 2 3 4 5
Detection of IS6110 gene in Mycobacterium tuberculosis
IC
IS6110
700
400
250
100
bp

16. Detection of PCR products by Capture Probes
Amount of signal is directly
proportional to the
amplicons present
E.g. viral load estimation

17. DNA sequencing
 Common method for analyzing amplified product
 Analysis of target generated amplicon – helps to
detect pathogen
 Helps to detect mutations in viruses / bacteria
 Genotyping of organism

18. TYPES OF PCR
 Qualitative PCR
 Quantitative PCR
 Nested PCR
 Multiplex PCR
 Reverse transcriptase PCR
 Real time PCR

19. TYPES OF PCR
 QUALITATIVE PCR: To detect the presence or
absence of a specific gene
 QUANTITATIVE PCR (qPCR): The amount of product
synthesized during a test PCR is compared with the
amounts synthesized during PCRs with known
quantities of starting DNA
Conventional method: Agarose gel electrophoresis
Real-Time PCR: Product is measured over time

20. NESTED PCR
•Two sets of primers
•First set of primer directed
against the target gene
•Second set of primers are
internal to the first set target
Types
Single step nested PCR
Two step nested PCR
Uses: HSV, CMV, Enterovirus
M.tuberculosis

21. Multiplex PCR
 Multiple primer sets for simultaneous detection of several
targets within a single PCR reaction
Clinical application
To detect microorganism causing single type of disease
 Meningitis
 Diarrhoeagenic E. coli
 Respiratory viruses
 Virulence genes of STEC
Disadvantages
Sensitivity is decreased when compared to uniplex PCR
Primers may cross react

22. Multiplex PCR for the detection of
Shiga toxigenic Escherichia coli
kb
1000
700
400
200
hly
eae
stx1
stx2
M 1 2 3 4 Pc Nc

23. Reverse transcriptase PCR (RT-PCR)
 RTase enzyme is used to synthesize DNA from RNA
Types
 Single step RT PCR
Single termostable DNA polymerase that also possess
significant RTase activity is used
 Two step RT PCR
Separate RTase enzyme is used prior to the addition of DNA
polymerase
Uses
 detection of RNA viruses
 detection of microbes – rRNA – viable
 to study gene expression
 quantitative RT PCR – viral load

24. REAL TIME PCR
 Synthesis of the amplicons is measured over time,
as the PCR proceeds through its series of cycles
 Amount of amplicons formed are directly
proportional to the target DNA in the starting
material
 It enables both detection and quantification of a
specific DNA sequence in the sample
 By using fluorescent dye or reporter molecule

25. REAL TIME PCR
 Nonspecific detector system
 SYBR Green gives fluorescent signal when it binds
to double-stranded DNA
 This method measures the total amount of double-
stranded DNA in the PCR
 Primer dimers

26. REAL TIME PCR - Specific detector system
 Reporter probe gives fluorescent signal when it hybridizes to
the PCR product - Specific
 Each reporter probe has pair of labels
 A fluorescent dye at one end of the oligonucleotide and a
quenching compound to the other end
 Two ends of oligonucleotides base pair to one another, placing
the quencher next to the dye – No fluorescence
 Hybridization between the oligonucleotide and the PCR product
disrupts base pairing quencher moves away from the dye and
fluorescent signal generated

27. Uses of Real time PCR
 Quantitation of gene expression
 Pathogen detection
 Viral quantitation
 Array verification
 Drug therapy efficacy
 DNA damage measurement
 Quality control and assay validation
Advantages of Real time PCR
1 Rapid
2 Quantitative measurement
3 Lower contamination rate
4 Higher sensitivity
5 Higher specificity
6 Easy standardization

28. Application of PCR in Clinical Microbiology
 Detection of slow-growing or fastidious microorganisms
 Detection of infectious agents that cannot be cultured
 Recognition of newly emerging pathogen
 Detection of RNA viruses
 Diagnosis of viral encephalitis
 Identification of Mycobacterium spp.
 Estimation of viral load to monitor therapy
 Detection of bacterial DNA for the diagnosis of septic arthritis
and reactive arthritis
 Identification of antimicrobial resistance genes

29. Advantages of PCR
 Speedy diagnosis
 Ease of use
 Sensitivity
 Specificity

30. Disadvantages of PCR
 Need for target DNA sequence information
 Primer Designing for unexplored ones
 Taq Pol – no Proof reading mech – Error 40% after 20
cycles
 Short size genes Up to 40 Kb can be amplified
 Technical expertise

31. THANK YOU