Quantitative �Real Time� PCR by 2rZL2o

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									PCR for Aquatic Animal Health

          Web Training Module V1; August 2011
             Created by Maureen Purcell, Ph.D.
Goal
   To provide an overview of PCR-based diagnostic
    assays with an emphasis on basic theory
    •   Want to learn more?
        •   Click on the reference links located at the bottom of certain
            slides
Content Overview
   PCR basics
   Commonly used PCR assays
   Advantages and disadvantages of PCR
   Good laboratory practices
   Analytical validation
   Sampling and template preparation
   Primers
   Standards, controls and normalization
   Quantitative PCR – in depth


Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
   PCR Basics
       Polymerase chain reaction (PCR) is a method to
        amplify a target sequence from background
        nucleic acid

PCR uses synthetic                                                   Target Sequence
                                           5’                                                                          3’
oligonucleotide primers
that flank target                        Forward Primer                                                 Reverse Primer
sequence




DNA synthesis is
                                                            T



catalyzed in vitro by a                                                                                  Reverse Primer
                                         Forward Primer
heat stable DNA
polymerase                                       Taq
                                             Polymerase


       Lodish H, A. et al.. (2000) Polymerase chain reaction, an alternative to cloning. In Molecular Cell Biology. 4th edition. W.H.
       Freeman, NY. Section 7.7. http://www.ncbi.nlm.nih.gov/books/NBK21541/
PCR Basics
    PCR basic steps
                                    5’                                                              3’
     Denature DNA
        (94°C)
                                    3’                                                              5’



                                    5’                                                              3’
    Anneal primer
                                Forward Primer                                           Reverse Primer
     (~50 = 65°C)
                                    3’                                                              5’




     Extension
                                                  T




                                                                                          Reverse Primer
      (72°C)                     Forward Primer




      http://www.idtdna.com/pages/docs/educational-resources/the-polymerase-chain-reaction.pdf
PCR Basics
    Stages of PCR


                                                                           Plateau Phase
                 Log Target




                                                                      Linear Phase

                                                              Exponential (Geometric) Phase

                                                            Stochastic/ ‘lag’ phase

                                         Cycle Number




    http://www6.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf
PCR Basics
   Theoretically the target sequence is doubled every
    PCR cycle
   This doubling each cycle equates to 100% PCR
    efficiency or an efficiency (E) of 2
                                           Theoretical
               Log Target




                            Cycle Number
PCR Basics
   In practice, PCR efficiency will vary depending
    on a range of factors



                                             Theoretical Efficiency (E) = 2
             Log Target DNA




                                                   Actual Efficiency (E) < 2




                              Cycle Number
Commonly Used PCR Assays
   Conventional PCR utilizes two primers and
    products are detected by gel electrophoresis
                   “cPCR”

                                                                               Agarose gel
                                                                             electrophoresis
                                                                              following PCR
                Log Target




                                       Cycle Number




    http://www.idtdna.com/pages/docs/educational-resources/gel-electrophoresis.pdf
Commonly Used PCR Assays
   A reverse-transcriptase step can be added to
    the PCR when the starting template is RNA
                   “RT-PCR”

The RT reaction can be primed by a:
    target specific primer (i.e. primer targeting VHSV nucleocapsid (N) gene)
    oligo dT primer (a primer consisting of a run of T’s that targets the mRNA
         poly A tail)
    random primers (a mix of 6 base primers consisting of random nucleotides)

                 All messenger RNAs (mRNA) have a poly A tail

                                   RNA                     AAAAAAAAAAAA

                                                          TTTTTT
                                                         oligo dT primer

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Amplification-and-
Expression-Profiling/Reverse-Transcription-and-cDNA-Synthesis/RNA-Priming-Strategies.html
Commonly Used PCR Assays
   A reverse-transcriptase step can be added to
    the PCR when the starting template is RNA
                   “RT-PCR”

RNA is copied into
complementary DNA                                         RNA                    AAAAAAAAAAAA
(cDNA) by the reverse
transcriptase enzyme                                     cDNA                    TTTTTT




http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Amplification-and-
Expression-Profiling/Reverse-Transcription-and-cDNA-Synthesis/RNA-Priming-Strategies.html
Commonly Used PCR Assays
   Some vendors sell “one-step RT-PCR” master mixes
      This is a misnomer and should be called one-
       tube RT-PCR
      RT-PCR always involves two steps
        1. Reverse-transcriptase
        2. PCR
            These steps can be performed in the same
              reaction tube (aka one-step) or in separate
              reaction tubes
Commonly Used PCR Assays
    Nested PCR (“nPCR”) involves two rounds of PCR
     utilizing outer and inner primer sets to improve
     sensitivity (because two rounds of PCR are
     performed) and specificity (since all four primers
     must match the target sequence)
                       Target DNA Region (i.e. Msa gene from R. salmoninarum)

Outer Forward Primer                                                         Outer Reverse Primer



                                       1st Round PCR Product

                       Inner Forward Primer                                  Inner Reverse Primer




                                                    2nd Round PCR Product



    http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/1.1.05._VALID_PCR.pdf
Commonly Used PCR Assays
    Real-time PCR detects a fluorescent signal that
     is increased each time a template is copied; the
     fluorescent signal is monitored each cycle or in
     ‘real-time’
                   ∆ Fluorescence




                                                                        Threshold



                                          CT CT
                                        Cycle Number

          CT = The cycle that a PCR reaction crosses the designated threshold
               Also called cycle quantification (CQ) or crossing point (CP)
    http://www6.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf
Commonly Used PCR Assays
   Quantitative PCR relies on the principal that
    the quantity of target at the start of the reaction
    is proportional to amount of product produced
    during the exponential phase


                           Greater starting target
          ∆ Fluorescence




                                                     Less starting target




                                       CT < CT
Commonly Used PCR Assays
   Real-time PCR is often used synonymously with
    quantitative PCR
     Real-time PCR involves monitoring the fluorescent
      signal produced during every cycle
     Real-time PCR results can be interpreted as plus
      or minus (detectable / not detectable)
      amplification
     Real-time PCR results can be used to estimate
      starting quantity of the target sequence in a
      sample = quantitative PCR
Commonly Used PCR Assays
   Suggested terminology and acronyms for each assay type

    Assay type                                Acronym      Nucleic acid        Result
                                                             target
    Detection by gel-based electrophoresis

    Conventional PCR                            cPCR           DNA          Plus / Minus

    Reverse transcriptase conventional PCR    RT-cPCR          RNA          Plus / Minus

    Nested PCR                                  nPCR           DNA          Plus / Minus

    Reverse transcriptase nested PCR          RT-nPCR          RNA          Plus / Minus



    Detection by fluorescent monitoring in a real-time PCR instrument

    Quantitative PCR                            qPCR           DNA        CT / Pathogen copy

    Reverse transcriptase quantitative PCR    RT-qPCR          RNA        CT / Pathogen copy

    Real-time PCR                               rPCR           DNA          Plus / Minus

    Reverse transcriptase real-time PCR       RT-rPCR          RNA          Plus / Minus
Advantages and Disadvantages of PCR
   Detection of pathogens with PCR-based tests
    have a number of general advantages
       Assays are typically highly sensitive
       Assays are typically highly specific
       Assays can be run in a high through-put manner




Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Advantages and Disadvantages of PCR
   Detection of pathogens with PCR-based tests
    have a number of general disadvantages
       Failure to detect pathogen template due to genetic variation
        at primer sites leading to false-negative results
       Inhibitors in samples leading to false-negative results
       High risk of contamination leading to false-positive results
       No indication of pathogen viability
       Confirms presence of nucleic acid but not infection
       Only a small proportion of the tissue is examined per
        reaction




Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Advantages and Disadvantages of PCR
   Nested PCR for pathogen
    detection                                                                              Bacterial Quantity
                                                                                             104 103 102 101
       Advantages
           Two rounds of PCR                                                                          - -
            improves sensitivity                                    Conventional
                                                                       PCR
           Two sets of primers
            improves specificity
       Disadvantages
           Prone to contamination
            from amplified PCR                                               Nested
                                                                              PCR                     + +
            products
           Time consuming to perform
            two PCR rounds

Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Advantages and Disadvantages of PCR
     Quantitative PCR has several advantages over
      conventional and nested PCR assays
       Obtain quantitative estimate of target
       Semi-automated
       Rapid results
       No handling of amplified DNA which limits potential
        laboratory contamination
       Some assays use an internal probe that provides added
        specificity
       Good assay parameters
          Large dynamic range
          Low inter-assay variation
          Highly reliable


    Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
    laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Good Laboratory Practices
    All PCR-based assays are prone to contamination
         Need dedicated spaces for different activities:


         Clean Room:                       Sample                      Nested PCR:              Dirty Room:
          storage and                  Preparation:                   handling of first       PCR amplification
      preparation of PCR              all samples and                   round PCR              and handling of
            reagents                 controls processed                  products             amplified products




                                                                        •Work flow in unidirectional -
                                         High Risk
                                        Templates:                       moving from clean to dirty
                                      plasmid DNA or
                                     synthetic controls
                                                                        •No exchange of equipment,
                                          at high
                                      concentrations                     materials or lab jackets


    Quality Assurance / Quality Control for the Fish and Wildlife Fish Health Laboratories:
    http://www.fws.gov/aah/PDF/QI-FWS%20AAHP%20QA%20Program.pdf
Analytical Validation
  Validation encompasses assay development, assay
optimization, analytical performance at the bench-top
scale, and diagnostic performance to establish the
fitness of a new diagnostic assay for its intended
purpose
 Important to evaluate properties of specificity,
sensitivity and repeatability for all diagnostic tests




http://www.oie.int/fileadmin/Home/eng/Health_standards/aahm/2010/1.1.2_VALID.pdf
Analytical Validation
   Definition of important terms
    Term                           Definition
    Fitness of purpose             The intended purpose of the assay


    Analytical sensitivity (ASe)   The minimum number of copies reliably detected by the assay

    Analytical specificity (ASp)   The degree to which the assay does not detect (amplify) other
                                   pathogens
    Limit of detection (LOD)       Another term to describe analytical sensitivity

    Repeatability                  Agreement between sample replicates, both within an assay run and
                                   between independent assay runs, when tested by the same laboratory

    Reproducibility                Agreement among test results when the same samples is tested by
                                   different laboratories
    Ruggedness                     Reproducibility of an assay using different reagent brands or batches
                                   and different equipment




http://www.oie.int/fileadmin/Home/eng/Health_standards/aahm/2010/1.1.2_VALID.pdf
Analytical Validation
   Analytical sensitivity (ASe) / limit of detection (LOD)
     Theoretically one copy of the target must be
      present in the reaction for PCR to occur but this
      copy number will not be reliably detected
     Samples at or below the LOD typically have poor
      repeatability
     Extending the assay cycle numbers well beyond
      the LOD may produce spurious results




http://www.oie.int/fileadmin/Home/eng/Health_standards/aahm/2010/1.1.2_VALID.pdf
Sampling and Template Preparation
    Sample acquisition represents the first source
     of experimental variability
         Laboratories need clear acceptance / rejection
          criteria for a sample
         Sample integrity must be maintained between
          collection, transport and receipt of sample
         Nucleic acid degrading solution (e.g. sodium
          hypochlorite or commercial product) should be used
          to clean non-disposable sampling tools and work
          spaces between samples
             Alcohol and/or flaming tools is not sufficient to prevent
              cross-contamination of samples


    Quality Assurance / Quality Control for the Fish and Wildlife Fish Health Laboratories:
    http://www.fws.gov/aah/PDF/QI-FWS%20AAHP%20QA%20Program.pdf
Sampling and Template Preparation
   Stabilizing nucleic acids
       RNA degrades rapidly and should be stabilized
        immediately
           Common stabilization methods for RNA
               Snap-freezing in liquid nitrogen
               RNA stabilizing solution (e.g. RNAlater®)
               Long-term storage at -80°C
       DNA is more stable but can degrade if not properly
        handled
           Common stabilization methods for DNA
               Freezing at -20°C or -80°C
               95% ethanol
               Drying on special filters (e.g. FTA® Cards)
Sampling and Template Preparation
   Important to be familiar with general principles of
    working with RNA:
     Avoid RNAses
     Always wear gloves when handling reagents or
      equipment that will be used in the RNA extraction
      and reverse transcription procedures
     RNAse-free water can be commercially purchased
      or nanopure water can be treated with diethyl
      pyrocarbonate (DEPC)




http://www.promega.com/~/media/files/resources/product%20guides/rna%20analysis%20notebook/workingwithrna
.ashx?la=en
Sampling and Template Preparation
    A variety of commercial kits exist to extract
     nucleic acids
    New extraction methodologies need to be
     evaluated to assess impact on assay sensitivity
    High throughput methods need careful
     evaluation to ensure that no cross-
     contamination occurs among samples
    Spectrophotometric analysis to obtain DNA
     concentration is useful for monitoring
     extraction efficiency


    http://www.nanodrop.com/Library/T009-NanoDrop%201000-&-NanoDrop%208000-Nucleic-Acid-Purity-Ratios.pdf
Primers
   A variety of commercial companies can
    synthesize oligonucleotide primers
   Primers typically arrive lyophilized, are
    rehydrated with nuclease-free water, and stored
    at -20°C
   ‘Dilution’ and ‘Resupension’ online calculators
    to assist in primer dilution
       http://www.idtdna.com/analyzer/Applications/DilutionCalc/
       http://www.idtdna.com/analyzer/Applications/resuspensioncalc/
Standards, controls and normalization
     Standard: a sample of a known concentration/copy
      number used to construct the standard curve
     Control: various samples that ensure the validity of
      positive and negative results
     Normalization: corrects for variation in template
      quantity and/or template quality

     Endogenous: target naturally present in sample
      (e.g. host gene)
     Exogenous: artificial target that is spiked into the
      sample

           *See reference below for in depth discussion
    Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
    laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization
     Standard: a sample of a known concentration/copy
      number used to construct the standard curve


     Standards are typically used when quantitative results are desired
                             = quantitative PCR




    Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
    laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization
   A good standard:
     Stable
     Mimics the biological target
     Can be accurately quantified
     New batches can be reliably produced
     Not a high contamination risk for the
      laboratory




Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization
   Standards for DNA targets
       Plasmid DNA containing PCR target
       Single-stranded oligodeoxynucleotides
       Quantified pathogen culture
           e.g. Bacterium quantified by FAT
           e.g. CFU or PFU quantified pathogen
           e.g. Purified parasite spores
   Standards for RNA target
       Same as above
       In vitro transcript generated from plasmid
        (synthesized using T3 or T7 RNA polymerase)

Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization
     Control: various samples that ensure the validity of
      positive and negative results


              Controls Distinguish:
        true positives and true negatives
                       from
       false positives and false negatives



    Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
    laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization
     Optimal set of controls:
       Processing positive control
        Control for false negatives and extraction efficiency

          Processing negative control
           Control for false positives (extraction contamination)

          PCR no template control
           Control for false positives (PCR contamination)

          Standards diluted to the detection limit
           Control for false negatives

          Internal positive control (IPC)
           Irrelevant template and primers that are added to the assay
           Detects assay inhibitors (leading to false negatives)
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization
     Normalization: corrects for variation in template
      quantity and/or template quality




                 Normalization is typically only
             performed when data are quantitative




    Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
    laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization
     External (Exogenous) Normalizing Variables
       Tissue weight extracted
       Nucleic acid concentration
     Internal (Endogenous) Normalizing Variables
       RNA: endogenous host gene (housekeeping
        gene)
       DNA: can be done but not common




Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization
     Normalizing to tissue weight
         Advantages
              Typical ‘Fish Health Units’
                e.g. CFU/g tissue  gene copies/g tissue

         Disadvantages
              Extraction efficiency may vary
           Does not detect degradation of sample or inhibitors
           
     Normalizing to nucleic acid concentration
       Advantages
          Independent of extraction efficiency
          Done correctly, can be fairly reliable
       Disadvantages
          Time consuming to quantify samples
          Accuracy of spectrophotometer
          Impact of contaminating nucleic acids
          Does not detect degradation of sample or inhibitors



    Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
    laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization
     Normalizing to endogenous host gene
       Not recommended because expression of the typical
        endogenous normalizing gene varies considerably
         Inappropriate in field samples to use as a measure of
          ‘RNA quantity’
         Results should not be used to ‘normalize’ pathogen copy
          number
       Amplification of a housekeeping gene can be used to assess
        RNA quality (i.e. as a ‘control’)




    Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
    laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization
     Recommendations for the use of controls, standards and normalization
     Category             Type                           Recommendation
     Standards            Standard curve                 Always recommended when quantitative results are
                                                         desired
                          Reference sample               Always recommended to include a minimum of one
                                                         positive reference sample per assay run
     Controls             Positive processing            Always recommended to verify nucleic acid extraction
                          sample                         effectiveness
                          Negative processing            Always recommended to detect contamination during
                          sample                         extraction process
                          No template control for        Always recommended on every assay run to detect
                          reaction                       contamination in reagents
                          Internal positive              Good practice for detecting false negative results if IPC
                          control (IPC)                  does not interfere with assay sensitivity

                          Amplification of               Good practice for ensuring nucleic acid integrity and
                          endogenous gene                troubleshooting
     Normalization        Exogenous                      Good practice to track tissue weight and nucleic acid
                          normalization variables        concentration; normalizing copy number to these variables
                                                         is dependent on goals
                          Normalization to               Not recommended to normalize copy number to
                          endogenous gene                endogenous gene expression in field samples


    Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
    laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Quantitative PCR – in depth
   Major assay types
       Fluorogenic 5’ Nuclease Assay
         Basis of TaqMan® chemistry
         Uses two primers and an internal hydrolysis probe
         Most commonly used for fish health diagnostics


       SYBR ® green dye chemistry
         Increased fluorescence when bound to dsDNA
         Slightly lower specificity
         Costs less
         May not be as sensitive as the 5’ nuclease assays




    http://www.clinical-virology.org/pdfs/PCR_experience.pdf
  Quantitative PCR – in depth
     Fluorogenic 5’ Nuclease Assay

          Step 1:                                                   Energy from fluorophore
                       Forward Primer     R                   Q     transferred to quencher
       Anneal and                                 Probe
      Polymerization
                                    Taq                                         Reverse Primer
                                              R
                                Polymerase
      Step 2:                                 T               Q
Strand Displacement


                                          R
      Step 3:                                                 Q
     Cleavage
  Polymerization
    Complete                  Probe must hybridize specifically for cleavage
                              A probe is cleaved each time a target is copied
Quantitative PCR – in depth
   Dual-labeled internal hydrolysis probes
     5’ reporter dye (typically Fam/Vic etc.)
     3’ quencher (typically non-fluorescent)
     Can order from a range of oligo companies
     Many companies have proprietary modifications for
      internal hydrolysis probes
        Minor Grove Binding (MGB) – Applied Biosystems Inc.
          The MGB linker raises the melting temperature of
           the internal hydrolysis probe and increases probe
           specificity




    http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_083
    618.pdf
Quantitative PCR – in depth
   Most common to use a commercial real-time PCR master mix
           Variety of vendors
           Variety of proprietary formulations
           Empirically evaluate how different formulations impact assay sensitivity
   Most master mixes contain:
           Passive normalizing dye to correct for variation in master mix
            concentration
           Hot-start Taq polymerase activation so reactions can be set-up at room
            temperature
           System to degrade post-PCR products
            Uracil-N-Glycosylase (UNG) degrades amplified products that have dUTP
Quantitative PCR – in depth
   Analysis of real-time PCR results are specific to the
    instrument
   Most instrument vendors provide training and
    technical support
Quantitative PCR – in depth
   Standards are needed if quantitative results are
    desired
   Standard curve that plots log copy number against
    cycle threshold (CT)

                   35
                   30                                y = -3.3169x + 38.322
                                                         R2 = 0.9989
                   25
                   20
              CT




                   15
                   10
                   5
                   0
                        0.0   2.0   4.0        6.0     8.0          10.0
                                      Log Copy #


            Quantity is determined by equation of the line
                        Antilog ((CT-y int)/m)
Quantitative PCR – in depth
   Reliable endpoint of assay should be defined
    empirically during assay validation
                40.0
                                   5 plasmid copies          y = -3.5689x + 38.561
                35.0                                               R2 = 0.99
                30.0
                25.0
           CT




                20.0
                15.0
                10.0
                 5.0
                 0.0
                       0.0   1.0      2.0      3.0     4.0         5.0         6.0
                                     Log (RS plasmid copies)


      Analytical sensitivity: the smallest number of genome copies
       that can be (reliably) detected and distinguished from zero
Quantitative PCR – in depth
   Low initial starting copy
    numbers impacts the
    accuracy and precision of
    quantitative PCR
       Statistical errors impact
        quantification when starting
        copy number is < 1000
   Results are not always
    reproducible beyond the            Random effects
                                          in PCR
    reliable endpoint of the assay
Acknowledgements
   Prepared by:
      Maureen Purcell
      Western Fisheries Research Center
      U.S. Geological Survey
      6505 NE 65th St, Seattle WA 98034
      mpurcell@usgs.gov


   The use of trade, firm, or corporation names in this publication is for
    the information and convenience of the reader. Such use does not
    constitute an official endorsement or approval by the U.S.
    Department of Interior or U.S. Geological Survey of any product or
    service to the exclusion of others that may be suitable.

								
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