pcr for aquatic animal health web training module v1; august 2011 created by maureen purcell, ph.d
TRANSCRIPT
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
Forward Primer Reverse Primer
PCR uses synthetic oligonucleotide primers that flank target sequence
TaqPolymerase
Target Sequence
DNA synthesis is catalyzed in vitro by a heat stable DNA polymerase
5’ 3’
T
Forward PrimerReverse Primer
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
Denature DNA (94°C)
Extension (72°C)
T
Forward PrimerReverse Primer
5’ 3’
3’ 5’
Anneal primer (~50 = 65°C)
5’ 3’
3’ 5’
Forward Primer Reverse Primer
http://www.idtdna.com/pages/docs/educational-resources/the-polymerase-chain-reaction.pdf
PCR Basics Stages of PCR
Cycle Number
Exponential (Geometric) Phase
Plateau Phase
Stochastic/ ‘lag’ phase
Log T
arg
et
Linear Phase
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 2Lo
g T
arg
et
Cycle Number
Theoretical
PCR Basics In practice, PCR efficiency will vary depending
on a range of factors
Log T
arg
et
DN
A
Cycle Number
Theoretical
Actual Efficiency (E) < 2
Efficiency (E) = 2
Commonly Used PCR Assays
Log T
arg
et
Agarose gel electrophoresi
s following PCR
Cycle Number
Conventional PCR utilizes two primers and products are detected by gel electrophoresis
“cPCR”
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)
RNA AAAAAAAAAAAA
TTTTTT
oligo dT primer
All messenger RNAs (mRNA) have a poly A tail
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 (cDNA) by the reverse transcriptase enzyme
RNA AAAAAAAAAAAA
TTTTTTcDNA
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-transcriptase2. 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)
Inner Forward Primer Inner Reverse Primer
1st Round PCR Product
Outer Forward Primer Outer Reverse Primer
2nd Round PCR Product
Target DNA Region (i.e. Msa gene from R. salmoninarum)
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’
∆ F
luore
scence
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
∆ F
luore
scence
CT CT
Greater starting target
Less starting target
<
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 target
Result
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 Advantages
Two rounds of PCR improves sensitivity
Two sets of primers improves specificity
Disadvantages Prone to contamination
from amplified PCR products
Time consuming to perform two PCR rounds
+ +
- -
Bacterial Quantity
Nested PCR
104 103 102 101
Conventional 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
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: storage and
preparation of PCR reagents
Sample Preparation: all samples and
controls processed
Dirty Room: PCR amplification and handling of
amplified products
Nested PCR: handling of first
round PCR products
High Risk Templates:
plasmid DNA or synthetic controls
at high concentrations
•Work flow in unidirectional - moving from clean to dirty
•No exchange of equipment, 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 DefinitionFitness 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 controlControl for false negatives and extraction efficiency
Processing negative controlControl for false positives (extraction contamination)
PCR no template controlControl for false positives (PCR contamination)
Standards diluted to the detection limitControl for false negatives
Internal positive control (IPC)Irrelevant template and primers that are added to the assayDetects 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 RecommendationStandards Standard curve Always recommended when quantitative results are
desiredReference sample Always recommended to include a minimum of one
positive reference sample per assay runControls Positive processing
sampleAlways recommended to verify nucleic acid extraction effectiveness
Negative processing sample
Always recommended to detect contamination during extraction process
No template control for reaction
Always recommended on every assay run to detect contamination in reagents
Internal positive control (IPC)
Amplification of endogenous gene
Good practice for detecting false negative results if IPC does not interfere with assay sensitivity
Good practice for ensuring nucleic acid integrity and troubleshooting
Normalization Exogenous normalization variables
Good practice to track tissue weight and nucleic acid concentration; normalizing copy number to these variables is dependent on goals
Normalization to endogenous gene
Not recommended to normalize copy number to 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
Forward Primer
Reverse Primer
Step 1:Anneal and
Polymerization
R QEnergy from fluorophore transferred to quencher
R
QStep 2:Strand Displacement
T
RQStep 3:
CleavagePolymerization
Complete Probe must hybridize specifically for cleavageA probe is cleaved each time a target is copied
Probe
TaqPolymerase
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_083618.pdf
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)
Quantitative PCR – in depth
y = -3.3169x + 38.322
R2 = 0.9989
0
5
10
15
20
25
30
35
0.0 2.0 4.0 6.0 8.0 10.0
Log Copy #
CT
Quantity is determined by equation of the lineAntilog ((CT-y int)/m)
y = -3.5689x + 38.561
R2 = 0.99
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0
Quantitative PCR – in depth
Analytical sensitivity: the smallest number of genome copies that can be (reliably) detected and distinguished
from zero
Log (RS plasmid copies)
CT
5 plasmid copies
Reliable endpoint of assay should be defined empirically during assay validation
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 reliable endpoint of the assay
Random effects in PCR
Acknowledgements Prepared by:
Maureen Purcell
Western Fisheries Research Center
U.S. Geological Survey
6505 NE 65th St, Seattle WA 98034
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.