Investigation strategies and methods

Polymerase Chain Reaction

May 2007

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T 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

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 History of PCR

Invented and patented in 1983

Revolutionary technique

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 PRC overview

Enzymatic DNA amplification

Need two short sequences on the DNA

Repetition of 30-35 cycles of three steps

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 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

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 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……..

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 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)

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 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)

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 What you need for PCR

Put into one reaction tube:

• Sample (+/- target DNA)

• Primers for the specific detection

• Nucleotides

• Enzyme

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 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

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 Logaritmic multiplication

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 Logaritmic multiplication

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 Logaritmic multiplication

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 Logaritmic multiplication

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 Logaritmic multiplication

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 Logaritmic multiplication

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 Logaritmic multiplication

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
Laboratory Training for FieldEEpidemiologists
P I D E M I C A L E R T A N D R E S P O N S E
 Advantages of PCR

Quick

Reliable

Sensitive

Relatively easy

Specific

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 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
Laboratory Training for FieldEEpidemiologists
P I D E M I C A L E R T A N D R E S P O N S E
 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
Laboratory Training for FieldEEpidemiologists
P I D E M I C A L E R T A N D R E S P O N S E
 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

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 PRC diagnostics

Viruses

• HIV, SARS, H5N1

Bacteria

• meningococcus, legionellosis

Analysis for resistant genes

• MRSA, VRE

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E
 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

Laboratory Training for FieldEEpidemiologists

P I D E M I C A L E R T A N D R E S P O N S E