E 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 Field Epidemiologists

Polymerase Chain Reaction

Investigation strategies and methods

May 2007

E 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 Field Epidemiologists

Learning objectivesLearning 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

E 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 Field Epidemiologists

History of PCRHistory of PCR

Invented and patented in 1983

Revolutionary technique

E 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 Field Epidemiologists

PRC overviewPRC overview

Enzymatic DNA amplification

Need two short sequences on the DNA

Repetition of 30-35 cycles of three steps

E 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 Field Epidemiologists

Technical overviewTechnical 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

E 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 Field Epidemiologists

BackgroundBackground

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……..

E 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 Field Epidemiologists

What you need for PCRWhat you need for PCR

Two short DNA fragment that stick specifically to eachof the DNA strands at some distance of each other

Primers

• Can be specific for:

– A certain bacterium

– Bacterial species

– Genes (e.g., toxin gene)

E 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 Field Epidemiologists

What you need for PCRWhat you need for PCR

Apparatus to perform about 35 cycles of a threetemperature procedure

• 95 °C (denaturation of DNA)

• 50-60 °C (annealing of primers)

• 72 °C (extension of the primers)

E 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 Field Epidemiologists

What you need for PCRWhat you need for PCR

Put into one reaction tube:

• Sample (+/- target DNA)

• Primers for the specific detection

• Nucleotides

• Enzyme

E 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 Field Epidemiologists

Performing PCRPerforming 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

E 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 Field Epidemiologists

Logaritmic multiplicationLogaritmic multiplication

E 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 Field Epidemiologists

Logaritmic multiplicationLogaritmic multiplication

E 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 Field Epidemiologists

Logaritmic multiplicationLogaritmic multiplication

E 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 Field Epidemiologists

Logaritmic multiplicationLogaritmic multiplication

E 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 Field Epidemiologists

Logaritmic multiplicationLogaritmic multiplication

E 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 Field Epidemiologists

Logaritmic multiplicationLogaritmic multiplication

E 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 Field Epidemiologists

Logaritmic multiplicationLogaritmic multiplication

E 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 Field Epidemiologists

E 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 Field Epidemiologists

Advantages of PCRAdvantages of PCR

Quick

Reliable

Sensitive

Relatively easy

Specific

E 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 Field Epidemiologists

Disadvantage of PCRDisadvantage of PCRNeed for equipment

Taq polymerase is expensive

Contamination

False reactions

Internal control

Cross-reaction

Enrichment steps in (contaminated) samples

Capacity building needed

Unspecific amplification

E 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 Field Epidemiologists

Applications of PCRApplications of PCRDetection 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 areoften repeated in the genome

E 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 Field Epidemiologists

Restrictions of PCRRestrictions of PCR

Contamination of reagents or lab results in falsepositive results

Failure due to a mistake in the protocol

Different materials/parts of the sample can inhibit thePRC process

E 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 Field Epidemiologists

PRC diagnosticsPRC diagnostics

Viruses

• HIV, SARS, H5N1

Bacteria

• meningococcus, legionellosis

Analysis for resistant genes

• MRSA, VRE

E 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 Field Epidemiologists

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

Investigation strategies and methods