Investigation strategies and methods
Polymerase Chain Reaction
May 2007
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E
Learning objectives
At the end of the presentation, participants should know:
•
History of polymerase chain reaction (PCR)
•
Definition and short technical overview of PCR
•
Applications of PCR
•
Restrictions of PCR
•
Examples for diagnostics with PCR
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Laboratory Training for FieldEEpidemiologists
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History of PCR
Invented and patented in 1983
Revolutionary technique
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Laboratory Training for FieldEEpidemiologists
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PRC overview
Enzymatic DNA amplification
Need two short sequences on the DNA
Repetition of 30-35 cycles of three steps
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Laboratory Training for FieldEEpidemiologists
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Technical overview
DNA consists of four elements: A, C, G and T
DNA molecule
• Double stranded DNA strands
• Bound together by chemical forces
– Exception: single stranded DNA/RNA viruses
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Laboratory Training for FieldEEpidemiologists
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Background
Double stranded DNA:
…….A T G G C A T A T C G……..
…….T A C C G T A T A G C……..
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Laboratory Training for FieldEEpidemiologists
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What you need for PCR
Two short DNA fragment that stick specifically to each
of the DNA strands at some distance of each other
Primers
• Can be specific for:
– A certain bacterium
– Bacterial species
– Genes (e.g., toxin gene)
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Laboratory Training for FieldEEpidemiologists
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What you need for PCR
Apparatus to perform about 35 cycles of a three
temperature procedure
• 95 °C (denaturation of DNA)
• 50-60 °C (annealing of primers)
• 72 °C (extension of the primers)
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Laboratory Training for FieldEEpidemiologists
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What you need for PCR
Put into one reaction tube:
• Sample (+/- target DNA)
• Primers for the specific detection
• Nucleotides
• Enzyme
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Laboratory Training for FieldEEpidemiologists
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Performing PCR
1.
Put your tube in the apparatus
2.
Let the program run (35 cycles)
3.
If primers fit, there is amplification of target DNA
4.
If primers do not fit, no amplification product
=> the DNA (micro-organism) was not in the sample
5.
Detect if there is PCR product
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E
Logaritmic multiplication
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E
Logaritmic multiplication
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E
Logaritmic multiplication
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E
Logaritmic multiplication
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E
Logaritmic multiplication
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E
Logaritmic multiplication
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E
Logaritmic multiplication
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
A N D
R E S P O N S E
Advantages of PCR
Quick
Reliable
Sensitive
Relatively easy
Specific
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Laboratory Training for FieldEEpidemiologists
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Disadvantage of PCR
Need for equipment
Taq polymerase is expensive
Contamination
False reactions
Internal control
Cross-reaction
Enrichment steps in (contaminated) samples
Capacity building needed
Unspecific amplification
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
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Applications of PCR
Detection of specific genome
• Classical with a primer pair
• Nested – amplification of larger area then specific detection
in multiplied genome part (more sensitive)
• Real time PCR to quantify the amount of genome in sample
• Detection of RNA with reverse transcriptase
Screening specific genes for unknown mutations
Genotyping using short primers or primer pairs that are
often repeated in the genome
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
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Restrictions of PCR
Contamination of reagents or lab results in false
positive results
Failure due to a mistake in the protocol
Different materials/parts of the sample can inhibit the
PRC process
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PRC diagnostics
Viruses
• HIV, SARS, H5N1
Bacteria
• meningococcus, legionellosis
Analysis for resistant genes
• MRSA, VRE
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Investigation strategies and methods
Developed by the Department of Epidemic and
Pandemic Alert and Response of the World Health
Organization with assistance from:
European Program for Intervention
Epidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
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