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 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 History of PCR Invented and patented in 1983 Revolutionary technique 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 PRC overview Enzymatic DNA amplification Need two short sequences on the DNA Repetition of 30-35 cycles of three steps 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 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 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 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…….. 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 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) 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 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) 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 What you need for PCR Put into one reaction tube: • Sample (+/- target DNA) • Primers for the specific detection • Nucleotides • Enzyme 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 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 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 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 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 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 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 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 PRC diagnostics Viruses • HIV, SARS, H5N1 Bacteria • meningococcus, legionellosis Analysis for resistant genes • MRSA, VRE 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 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 A N D R E S P O N S E