pcr for aquatic animal health web training module v1; august 2011 created by maureen purcell, ph.d

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


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


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


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


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


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


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


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


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


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)


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


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)


Standards, controls and normalization Normalization: corrects for variation in template

quantity and/or template quality

Normalization is typically only performed when data are quantitative


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


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


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’)


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


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


Standards are needed if quantitative results are desired

Standard curve that plots log copy number against cycle threshold (CT)


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


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

[email protected]

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.

pcr for aquatic animal health web training module v1; august 2011 created by maureen purcell, ph.d

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