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An introduction to real-time PCR             and its applications in         food safety and environmental                ...
The needs of food safety testing facilitiesThe globalized food market calls for more stringent safety and quality testing ...
The needs of environmental testing facilities.   Public safety demands rapid and accurate safety and quality testing for w...
Advantages of real-time PCR Delivers results in approximately 3 hours Accurately detects DNA in challenging samples Has...
PCR-based testing with QIAGEN’s mericon portfolioAn integrated testing system that meets laboratory demands.     Sample p...
PCR-based testing with QIAGEN’s mericon portfolioCoverage.     Broad range of sample types     Success with challenging ...
PCR workflow for food safety testing                       -7-             Sample & Assay Technologies
Comparison of workflows for food pathogen detectionQIAGEN’s mericon workflow yields rapid, sensitive and highly specific r...
Introduction to real-time PCRReal-time versus traditional PCR.     Real-time PCR allows detection during early stages of ...
PCR components    PCR= Polymerase Chain Reaction    Exponential amplification of DNA in single tube                       ...
PCR in action                                                 DNA template                                                ...
PCR in action             Denature template                                                      DNA template             ...
PCR in action                                            DNA Template                                            (ss or ds...
PCR in action             Polymerase                          Polymerase                                                  ...
PCR in action                       Polymerase        Polymerase                                                    DNA te...
PCR in action                                                      Polymerase                                             ...
PCR in action                                            Polymerase                                     DNA template      ...
Advantages of real-time PCR Real-time PCR is a highly sensitive and reliable method for the   detection and quantificatio...
Real-time PCR chemistriesThere are many real-time chemistries available..     Intercalating dyes:         SYBR Green I  ...
Hydrolysis-based probe                   The fluorescence of the reporter dye                   is suppressed by the quenc...
How does a real-time PCR cycler work?  Thermal cycler                                                                     ...
Rotor-Gene Q — superior rotary optics                Reaction chamber                                          Detection  ...
Rapid air based cycling — heatingHeating mechanism                                                              Heater    ...
Rapid air based cycling — coolingCooling mechanism                                                                    Heat...
Real-time PCR terminologyThese terms are important to the analysis of real-time PCR data:.    Baseline    Threshold    ...
Baseline           Baseline           The initial cycles prior to amplification in which there is           little change ...
Threshold     Threshold     The level at which the amplification     fluorescence is above the baseline, but the     react...
Threshold cycle (CT)     Threshold cycle (Ct)     The cycle number at which the amplification plot crosses the threshold  ...
No-Template Control (NTC)       No-Template Control (NTC)       There is no nucleic acid template in the reaction. This is...
Critical factors for a successful assay DNA or RNA sample preparation — template quality   Choose appropriate sample prep...
Effect of inhibitors   Increasing amounts of inhibitors can completely inhibit PCR            SDS       Phenol       EtOH ...
Inhibitor tolerance of mericon PCR assay                                                      .   Observed effects in PCR ...
QIAGEN Internal Control                         QIAGEN Internal Control                                  IC               ...
QIAGEN solutions for food and environmental testing                                                                       ...
QIAGEN workflow — sample preparation.               Food or environmental testing laboratory                     Suitable ...
QIAGEN workflow — assay setup and detection         +DNA             Suitable mericon assay*                         Assay...
Thermal cycling program setup for real-time PCR                      - 37 -         Sample & Assay Technologies
Real-time PCR results                                 Highly sensitive pathogen detection, even in                        ...
Proven for water testing applications                                         QIAGEN real-time enzyme kits                ...
Legionella pneumophila detectionThe assay detects DNA of Legionella pneumophila over a concentration range from10 million ...
Salmonella spp. detectionThe assay detects DNA of the genus Salmonella over a wide concentration rangedown to 10 copies pe...
Detection of salmonella in chocolate matrices          The QIAsymphony RGQ Pathogen Protocol.    Chocolate with coffee fil...
Complete food and environmental testing solutions                   QIAGEN provides                    Easy-to-use system...
Summary Real-time PCR is a rapid, sensitive, and reliable method  for the detection of pathogens in food and environmenta...
Thank youFor up-to-date licensing information and product-specific disclaimers, seethe respective QIAGEN kit handbook or u...
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Intro to real time PCR in food safety and environment

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An introduction to real-time PCR and its applications in food safety and environmental testing.
For further details on the described technology please visit www.qiagen.com

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  1. 1. An introduction to real-time PCR and its applications in food safety and environmental testing QIAGEN Dr. Marcia Armstrong Scientific Affairs Manager Food Safety Testing Group QIAGEN webinar. January 19, 2012-1- Sample & Assay Technologies
  2. 2. The needs of food safety testing facilitiesThe globalized food market calls for more stringent safety and quality testing  Pathogen detection  All major food pathogens, e.g., Salmonella spp., Listeria spp., E. coli  Genetically modified organism DNA detection  DNA introduced into plants for pesticide qualities or herbicide resistance  Ingredient authentication  For halal or kosher certification and to rule out cheap adulterantsTesting requirements.  Rapid — quick release of product to market  Specific and sensitive — no false positive or false negative results  Easy to use — straightforward adoption by laboratories -2- Sample & Assay Technologies
  3. 3. The needs of environmental testing facilities. Public safety demands rapid and accurate safety and quality testing for water, soil, air, etc.  Microbial pathogen detection, quantification, and genotype identification  Bacteria, viruses, fungi, and protozoa must all be identifiedTesting requirements.  Rapid — limiting danger to the public  Specific and sensitive — no false positive or false negative results  Easy to use — straightforward adoption by laboratories -3- Sample & Assay Technologies
  4. 4. Advantages of real-time PCR Delivers results in approximately 3 hours Accurately detects DNA in challenging samples Has simple protocols that can be fully automated Detects specific targets without false negatives and false positives Unaffected by food processing or high contaminant contents Real-time PCR is highly suitable for sensitive detection of pathogens in samples of food, animal feed, water, soil, and air. Real-time PCR is highly suitable for the specific DNA detection needed for GMO detection and ingredient authentication. -4- Sample & Assay Technologies
  5. 5. PCR-based testing with QIAGEN’s mericon portfolioAn integrated testing system that meets laboratory demands.  Sample preparation and detection assay kits and instrumentsTop performance.  Sensitive, target-specific, inhibitor-resistant chemistryEase of use.  Streamlined, robust, user-friendly sample preparation and assay protocolsSpeed.  Hands-off, automatable sample preparation  Rapid results with low- and high-throughput solutions -5- Sample & Assay Technologies
  6. 6. PCR-based testing with QIAGEN’s mericon portfolioCoverage.  Broad range of sample types  Success with challenging samples  Broad coverage of food and environmental pathogens  Broad range of GMO- and ingredient-specific assaysStandardization.  Streamlined, standardized sample preparation  Single consistent protocol for all assays -6- Sample & Assay Technologies
  7. 7. PCR workflow for food safety testing -7- Sample & Assay Technologies
  8. 8. Comparison of workflows for food pathogen detectionQIAGEN’s mericon workflow yields rapid, sensitive and highly specific results Real-time PCR with QIAGEN’s mericon workflow Immunoassay Traditional culture -8- Sample & Assay Technologies
  9. 9. Introduction to real-time PCRReal-time versus traditional PCR.  Real-time PCR allows detection during early stages of amplification  Detection during the exponential phase of amplification is very accurate  Traditional PCR uses agarose gels for detection  The results are not very accurate or discriminatory 10 copies 50 copies Agarose gel cannot discriminate between 10 copies 10 and 50 starting copies of DNAReal-time PCR.  Sequence amplification and real-time detection using sequence-specific probes  Rapid, sensitive, and specific detection of pathogens in biological samples -9- Sample & Assay Technologies
  10. 10. PCR components PCR= Polymerase Chain Reaction Exponential amplification of DNA in single tube DNA template (ss or ds)Polymerase Thermostable: can withstand temperatures up to ~95oC All reagents in dNTPs excess (non-limiting) Primers (2) - 10 - Sample & Assay Technologies
  11. 11. PCR in action DNA template (ss or ds)Polymerase dNTPs 1. Denature template (~95oC) 2. Anneal primer (~60oC) Primers (2) 3. Extend primer (~72oC) 4. Repeat (~95oC) - 11 - Sample & Assay Technologies
  12. 12. PCR in action Denature template DNA template (ss or ds)Polymerase dNTPs 1. Denature template (~95oC) 2. Anneal primer (~60oC) Primers (2) 3. Extend primer (~72oC) 4. Repeat (~95oC) - 12 - Sample & Assay Technologies
  13. 13. PCR in action DNA Template (ss or ds)Polymerase dNTPs 1. Denature template (~95oC) 2. Anneal primer (~60oC) 3. Extend primer (~72oC) 4. Repeat (~95oC) - 13 - Sample & Assay Technologies
  14. 14. PCR in action Polymerase Polymerase DNA Template (ss or ds)Polymerase dNTPs. 1. Denature template (~95oC) 2. Anneal primer (~60oC) 3. Extend primer (~72oC) 4. Repeat (~95oC) - 14 - Sample & Assay Technologies
  15. 15. PCR in action Polymerase Polymerase DNA template (ss or ds)Polymerase dNTPs 1. Denature template (~95oC) 2. Anneal primer (~60oC) 3. Extend primer (~72oC) 4. Repeat (~95oC) - 15 - Sample & Assay Technologies
  16. 16. PCR in action Polymerase DNA template (ss or ds) PolymerasePolymerase dNTPs 1. Denature template (~95oC) 2. Anneal primer (~60oC) 3. Extend primer (~72oC) 4. Repeat (~95oC) - 16 - Sample & Assay Technologies
  17. 17. PCR in action Polymerase DNA template (ss or ds) dNTPs 1. Denature template (~95oC) 2. Anneal primer (~60oC) 3. Extend primer (~72oC) 4. Repeat (~95oC) - 17 - Sample & Assay Technologies
  18. 18. Advantages of real-time PCR Real-time PCR is a highly sensitive and reliable method for the detection and quantification of nucleic acids (DNA, RNA, and cDNA). It is based on detection of fluorescence emitted from a reporter molecule in real time. This detection occurs during the accumulation of the PCR product with each cycle of amplification. This allows monitoring of the PCR reaction during early exponential phase, when the first significant increase in the amount of PCR product correlates to the initial amount of target template. - 18 - Sample & Assay Technologies
  19. 19. Real-time PCR chemistriesThere are many real-time chemistries available..  Intercalating dyes:  SYBR Green I  LC Green HRM Dyes  EvaGreen  SYTO 9  Probe-based chemistries:  TaqMan (Applied Biosystems/LTI)  FRET/Hybridization/LightCycler Probes (Roche)  Molecular Beacons  Linear probes - 19 - Sample & Assay Technologies
  20. 20. Hydrolysis-based probe The fluorescence of the reporter dye is suppressed by the quencher Primer binding is followed by extension Probe cleavage by Taq to free the reporter dye so the fluorescence intensity correlates with the initial sample input amounts. Taq has 5’→ 3’ exonuclease activity Each amplicon needs a sequence-specific probe - 20 - Sample & Assay Technologies
  21. 21. How does a real-time PCR cycler work? Thermal cycler More focusing Excitation filters, lenses, source and mirrorsExcitation Sources: Focusing filters,• Halogen Lamp lenses, and mirrors Detection device• Light Emitting Diode (LED)• Argon Ion Laser Raw data Detection Devices: • Charge Couple Device (CCD Camera) • Photomultiplier Tube (PMT) * Rotor-Gene Q • Photodiode - 21 - Sample & Assay Technologies
  22. 22. Rotor-Gene Q — superior rotary optics Reaction chamber Detection filters Lens PMT detector assembly Tubes in rotor LED light spin past optics source assembly Spindle/motor assembly - 22 - Sample & Assay Technologies
  23. 23. Rapid air based cycling — heatingHeating mechanism Heater Centrifugal fan drives elements air around chamber switch on Chamber vent seals to contain air Holes in the rotor allow free airflow - 23 - Sample & Assay Technologies
  24. 24. Rapid air based cycling — coolingCooling mechanism Heater Centrifugal fan drives elements air around chamber switch off Chamber vent opens expelling hot air Centrifugal fan Drives air into chamber Holes in the rotor allow free airflow Cool air in - 24 - Sample & Assay Technologies
  25. 25. Real-time PCR terminologyThese terms are important to the analysis of real-time PCR data:.  Baseline  Threshold  CT  No-Template Control (NTC)Let’s examine each of these terms a little bit more closely.. - 25 - Sample & Assay Technologies
  26. 26. Baseline Baseline The initial cycles prior to amplification in which there is little change in fluorescent signal. Typically between cycles 3-15 CT - 26 - Sample & Assay Technologies
  27. 27. Threshold Threshold The level at which the amplification fluorescence is above the baseline, but the reactions are still in the exponential phase. CT - 27 - Sample & Assay Technologies
  28. 28. Threshold cycle (CT) Threshold cycle (Ct) The cycle number at which the amplification plot crosses the threshold line. Roche instruments use the term crossing point (Cp). Ct - 28 - Sample & Assay Technologies
  29. 29. No-Template Control (NTC) No-Template Control (NTC) There is no nucleic acid template in the reaction. This is used to measure if there is any contamination of reaction components or non-specific primer-primer or primer-probe interactions. The fluorescent signal should be flat. Ct - 29 - Sample & Assay Technologies
  30. 30. Critical factors for a successful assay DNA or RNA sample preparation — template quality Choose appropriate sample preparation kits or reagents Inhibitors can compromise your PCR Assay design — specificity, efficiency, chemistry Consider your throughput and cost per result Use thoroughly validated assays Running PCR Choose high quality reagents (primer, probe, master mix) Data analysis tool Work with user-friendly, streamlined data analysis modules - 30 - Sample & Assay Technologies
  31. 31. Effect of inhibitors Increasing amounts of inhibitors can completely inhibit PCR SDS Phenol EtOH NaAc NaCl EDTA - - - - - - - 31 - Sample & Assay Technologies
  32. 32. Inhibitor tolerance of mericon PCR assay . Observed effects in PCR analysesInhibitor tolerance of multiplex PCR master mix  Effects on general CT range  Quenching of end fluorescence  Scattering of end fluorescence  Effects of buffer pH  Scattering of replicates  PCR boost mericon PCR Master Mix  Stable with most inhibitors  Less replicate scattering  No end fluorescence effects mericon assay . Assay from other supplier - 32 - Sample & Assay Technologies
  33. 33. QIAGEN Internal Control QIAGEN Internal Control IC AmplificationSample DNA/RNA Valid result  control Extraction PCR/RT-PCR QIAGEN Internal Control IC (High conc.)Sample DNA/RNA Valid result  Extraction and amplification control Extraction PCR/RT-PCR Flexible use — control of extraction and/or amplification - 33 - Sample & Assay Technologies
  34. 34. QIAGEN solutions for food and environmental testing Reaction Detection Disruption Purification Detection setup Target Detected nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars, Lipids, Salts, Urea, Carbonic acids, Cofactors, Precursors, Hemoglobins, Erythrocytes, Monocytes, Smooth endoplasmatic reticulum, Macrophages, Thrombocytes, Platelets, Lymphocytes, Basophils, Eosinophils, Neutrophils, Megacaryocytes, Plasma, Clotting factors, Actin, Microfilaments, Serum, Fibrin, Lysosomes, Ezrin, DNA, Hemaglobins, Heptaglobins, Transferrin, Fibrinogen, Serum albumin, tRNA, Salts, Polymerases, Centrioles, DNA DNA Information Immunoglobulins, Carrier proteins, Cytokines, Angiotensins, Chemokines, Bradykines, Plasma membranes, Ribosomes, Actin, Vesicles, Complement components, Nuclei, Rough endoplasmatic reticulum, Nucleoli, Golgi apparatus, Glycoproteins, Microtubules, Mitochondria, Mitochondrial nucleic acids, Vacuoles, Talin, Nucleolus, Polymerases, Ceramides, Chromosomes, Chromatin, mRNA, Cytoplasm, Leucocytes, Sugars,Disruption and thermal Manual or automated Manual or automated Detection of DNA or enzymatic lysis purification setup of PCR - 34 - Sample & Assay Technologies
  35. 35. QIAGEN workflow — sample preparation. Food or environmental testing laboratory Suitable DNA extraction kit, such as the mericon DNA Bacteria or Bacteria Plus Kit, Sample DNeasy mericon Food Kit, DNA or QIAamp UCP Pathogen Mini Kit - 35 - Sample & Assay Technologies
  36. 36. QIAGEN workflow — assay setup and detection +DNA Suitable mericon assay* Assay setup PCR Results* Assays are available for a broad range of specific pathogens, genetically modified organisms, and ingredients. QIAGEN also haskits suitable for lab-developed assays. - 36 - Sample & Assay Technologies
  37. 37. Thermal cycling program setup for real-time PCR - 37 - Sample & Assay Technologies
  38. 38. Real-time PCR results Highly sensitive pathogen detection, even in difficult food matrices such as peanut butter. Enrichment cultures of salmonella in buffered peptone water were prepared. DNA was extracted from serial dilutions of this enrichment culture. The mericon Salmonella spp Kit was used for the assay, and salmonella was reliably detected at a dilution factor of 1:100,000. Reliable detection of trace amounts of pig DNA. The mericon Pig Kit was used to test for pig DNA in a series of diluted samples. High amounts of DNA do not interfere with the PCR. The assay is sensitive enough to detect fewer than 10 copies of target DNA. - 38 - Sample & Assay Technologies
  39. 39. Proven for water testing applications QIAGEN real-time enzyme kits Highly sensitive detection of RNA and DNA  Many citations for water testing applications available Highly flexible  Can be used on any cycler Convenient setup  No need to optimize reaction and cycling conditions 2 Examples of pathogen nucleic acids detected withSensitive detection of Norovirus using the QuantTect Probe PCR and RT-PCR Kitsthe QuantiFast Pathogen RT-PCR +ICKit on the Rotor-Gene Q. Echovirus, Norovirus and somatic coliphages Enterococcus spp., Clostridium spp., Giardia spp., Salmonella spp. and bacteriophages Legionella spp., Cryptosporidium spp. - 39 - Sample & Assay Technologies
  40. 40. Legionella pneumophila detectionThe assay detects DNA of Legionella pneumophila over a concentration range from10 million down to 10 copies per reaction.  All assay targets gave a signal in the green fluorescent channel  The internal control (IC) was detected in the yellow fluorescent channel 0.5 0.6 0.45 IC 0.5 107 copies/reaction 106 copies/reaction 0.4 0.4 Delta Rn 105 copies/reaction 0.3 0.2 0.35 0.1 104 copies/reaction 0.3 0 103 copies/reaction 102 copies/reaction Delta Rn 0 5 10 15 20 25 30 35 40 Cycle numb er 0.25 101 copies/reaction NTC 0.2 0.15 0.1 0.05 0 0 5 10 15 20 25 30 35 40 Cycle number - 40 - Sample & Assay Technologies
  41. 41. Salmonella spp. detectionThe assay detects DNA of the genus Salmonella over a wide concentration rangedown to 10 copies per reaction.  All assays targets gave a signal in the green fluorescent channel  The internal control (IC) was detected in the yellow fluorescent channel 0.5 IC 0.6 0.45 0.5 104 copies/reaction 0.4 0.4 103 copies/reaction Delta Rn 0.3 102 copies/reaction 0.35 0.2 101 copies/reaction 0.1 NTC 0.3 0 Delta Rn 0 5 10 15 20 25 30 35 40 45 Cycle number 0.25 0.2 0.15 0.1 0.05 0 0 5 10 15 20 25 30 35 40 Cycle number - 41 - Sample & Assay Technologies
  42. 42. Detection of salmonella in chocolate matrices The QIAsymphony RGQ Pathogen Protocol. Chocolate with coffee filling Milk chocolate Dilution series Dilution series 1:10 1:10 1:100 1:100 1:1000 1:1000 1:10000 1:10000 Chocolate with coffee filling Milk chocolate Internal control dilution series Internal control dilution series - 42 - Sample & Assay Technologies
  43. 43. Complete food and environmental testing solutions QIAGEN provides  Easy-to-use systems that are fast to learn and implement with basic training in every lab  Large choice of validated workflows, depending on throughput and regulation  Access to R&D and application labs for specific development requests  Specific technical support in English and in local languages  Field service engineers, familiar with routine testing lab requirements  Extensive experience in molecular testing - 43 - Sample & Assay Technologies
  44. 44. Summary Real-time PCR is a rapid, sensitive, and reliable method for the detection of pathogens in food and environmental samples. Real-time PCR is highly suitable for the specific DNA detection needed for GMO detection and ingredient authentication. QIAGEN is here to support you in your food safety and environmental testing work. - 44 - Sample & Assay Technologies
  45. 45. Thank youFor up-to-date licensing information and product-specific disclaimers, seethe respective QIAGEN kit handbook or user manual. QIAGEN kithandbooks and user manuals are available at www.qiagen.com or can berequested from QIAGEN Technical Services or your local distributor. - 45 - Sample & Assay Technologies
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