Loop-Mediated Isothermal Amplification (LAMP) · 1. Amplification takes place at a single...
Transcript of Loop-Mediated Isothermal Amplification (LAMP) · 1. Amplification takes place at a single...
Loop-Mediated Isothermal Amplification (LAMP): Assay Development Challenges and Solutions
Agenda1. An introduction to LAMP
2. LAMP vs. PCR
3. Detection technologies for LAMP
4. Assay optimization
5. Assay troubleshooting
6. OmniAmp and LavaLAMP
7. Conclusions
How much experience do you have with LAMP assays?
First Question for You!
Introduction to LAMP and Comparison to PCR
1. Amplification takes place at a single temperature 2. Uses a polymerase with high strand displacement activity 3. Amplification is rapid 4. Can be used for RNA templates by addition of reverse transcriptase
(RT) or by using an enzyme with both RT and DNA pol activities
LAMP: An Isothermal Amplification Technology
1. Affordable2. Sensitive3. Specific4. User friendly5. Robust and rapid6. Equipment free7. Deliverable to end user
Advantage of LAMP: ASSURED
• LAMP is an ideal amplification technology for Point of Care/Point of Need assays
• Test platform using LAMP could fulfil WHO’s recommended ASSURED characteristics:
Which applications are you interested in performing using
LAMP assays? (choose all that apply)
Next Question
FIP (Forward Inner Primer) BIP (Backward Inner Primer)F3 (Forward Outer Primer) B3 (Backward Outer Primer)FL (Forward Loop Primer) BL (Backward Loop Primer)
LAMP PrimersA Six Primer Mix Produces Optimal LAMP Results
Figure: http://loopamp.eiken.co.jp/e/lamp/principle.html
Amplification IntermediatesGeneration of Key Stem Loop DNA Structure
Non-cyclical Steps
Figure: http://loopamp.eiken.co.jp/e/lamp/principle.html
Amplification ProductsGeneration of Multimeric DNAs with Inverted Repeats During Cycling Amplification
Cycling Amplification Steps
Figure: http://loopamp.eiken.co.jp/e/lamp/principle.html
LAMP WorkflowEasy with Low Complexity Instrumentation
Sample
Heat lysis/NA extraction
Amplification
Detection
5-15 min
≤30 min
Real time or end point
LAMP vs. PCRLAMP Assays are Faster, Simpler Once Developed
PCR LAMP
1. Requires temperature cycling Isothermal – single temperature
2. Requires 2 primers Requires 6 primers
3. Slow: Typically >1hr Rapid: Typically <30 min
4. Typical yield ~ 0.2 µg Typical yield ~ 10–20 µg
5. Not amenable to visual detection Amenable to visual detection based on turbidity etc.
6. Sensitive to sample matrix inhibitors Tolerant to sample matrix inhibitors
7. Can be multiplexed Difficult to multiplex
Detection Technologies
LAMP Amplification Detection MethodsMultiple Choices Are Available Depending on Needs
Mg Precipitation Colorimetric Agarose Gel Real Time Turbidity
End Point Analysis is Not Suitable for Optimization Experiments
2.5E
+07
2.5E
+06
2.5E
+05
2.5E
+04
2.5E
+03
2.5E
+02
2.5E
+01
2.5E
+00
NTC
1 kb
L
1 kb
L
Target Amount
DNA copies /reaction
Detection by Real Time Fluorescence & TTRA Quantitative Measure of Assay Performance
Time to Threshold (TTT)orTime to Result (TTR) provides a quantitative measure of assay performance
Signal Threshold
TTR Provides a Quantitative Parameter for Assay Optimization
Target Amount: NTC
Agarose Gel Visualization
Fluorescence TTR Analysis
More quantitative results
What type of nucleic acid targets do you want to detect using LAMP
assays? (choose all that apply)
Third Question
Assay Optimization
Goals for OptimizationFaster TTR and Lower Background Signals
• Increased reaction speed (faster TTR)
• Decreased non-specific amplification (slower TTR from Negative Control)
• Increased separation between Positive and Negative TTR
• Improved sensitivity (Detect low copy inputs)
Factors for OptimizationMultiple Parameters Should be Assessed
• Primers (design, concentration and ratio)
• Enzyme (concentration)
• Reaction temperature
• Mg ion concentration
• Reaction pH
• Additives such as betaine
Figure: http://loopamp.eiken.co.jp/e/lamp/principle.html
Designing and Choosing the Best Primer SetPrimer Design Dramatically Affects LAMP Quality
Seven different primer designs were tested at multiple temperatures
Illustrating the Effects of Primer Design on Assay Performance
Winners! Winners!
Additional Characterization of Primer Design #1Sensitive with Undetectable Background
Testing Primer Set #1 with Varying Target Amounts
Illustrating the Effect of Primer ConcentrationMaximizing the ∆TTR Between Positive & NTC
1X Primer Concentrations
FIP & BIP: 1.6 µM ea.FL & BL: 0.8 µM ea.F3 & B3: 0.2 µM ea.
C. difficile target was assayed at various primer concentrations
Best
Effect of Enzyme ConcentrationOptimizing Enzyme Amount Improves Performance
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0.2X 0.4X 0.6X 0.8X 1.0X 1.2X 1.4X 1.6X 1.8X
Aver
age
TTR
(min
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Enzyme Concentration
C. difficile target was assayed with varying enzyme concentrations (0.2X to 1.8X)
Plus Target (+):
NTC (-):
Illustrating the Effects of Enzyme Concentration (Amount) on Assay Performance
Illustrating the Effects of Temperature on Assay Performance
Effect of Assay TemperatureMajor Differences Based on Assay Temperature
DNA LAMP using M13 phage as target
Illustrating the Effects of Mg2+ Concentration on Assay Performance
Decreasing the Mg Ion Concentration Reduces Background Amplification
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A: 4 mM B: 6 mM C: 8 mM D: 10 mM
RT LAMP was carried out using MS2 target at different Mg concentrations
High Target #:
Medium Target #
No Target Control #:
Mg2+ Concentration
Tim
e to
Res
ults
(TTR
)
Illustrating the Effects of Buffer (Reaction) pH on Assay Performance
Reaction pH Has Less of an Effect on Positive TTR
PositiveNo target control
Reaction pH Affects Background (Negative) Amplification
pH 8.0
pH 8.8
C. difficile genomic DNA assayed at various buffer/reaction pHPositive - RedNegative - Black
No Target Control Reactions
pH 7
.8
pH 7
.9
pH 8
.0
pH 8
.1
pH 8
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pH 8
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pH 8
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pH 8
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pH 8
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pH 8
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pH 8
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100b
p L
Inclusion of Betaine May Improve Assay Performance
C. difficile Target
Positive:
Negative:
Improved ∆TTR
Use of DOE May Improve Optimization
Set objective(Reduce positive
TTR, increase negative TTR)
Select input factors([MgSO4], [KCl],
[(NH4)2SO4)] etc.)
Select output response
(TTR)
Select design(Screening design, response surface
design etc.)
Develop strategy(Target level,
replicates etc.)
Run experiments(Realtime to derive
TTR)
Analyze dataFit model
Interpret model
Confirm model
Detecting C. difficile Target in Untreated and Heated Stool Samples
Sample Quality Affects Assay PerformanceCrude Samples May Require Pre-treatment
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High Medium Low NTC
Tim
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min
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Target Level
Untreated Stool Sample:
Preheated Stool Sample:
Stool sample was either added directly to the LAMP reactions or preheated at 90°C for 5 min and then added to the LAMP reactions.
Assay Troubleshooting
TroubleshootingExamine the Optimization Parameters
• Primer design
• Primer purity
• Sample matrix inhibition
• Suboptimal reagent conc. (Enzyme, Mg, salt, primer etc.)
• Suboptimal assay condition (temperature, pH etc.)
• Target contamination
Solutions to LAMP Challenges: OmniAmp® and LavaLAMPTM
Thermostable OmniAmp® PolymeraseA LAMP Enzyme with RT and DNA Pol Activity
• Innate reverse transcriptase and DNA polymerase activity • Innate strand displacement activity
OmniAmp® Polymerase Ideal for MDx Point of Care and Available Now
Property OmniAmp®
Polymerase
Thermostability > 70°C +Strand displacement +Single enzyme based RNA or DNA detection +Isothermal amplification +Can be stably dried +No extraction required +
• Well-suited to DNA and RT-LAMP assay optimization• Increased specificity and flexibility (target and reaction conditions)• http://www.lucigen.com/OmniAmp-RNA-and-DNA-LAMP-Kit/
LavaLAMPTM, a 2X LAMP Master MixComing Soon!
Convenient 2X master mix formulation
Sensitive, specific and rapid detection of DNA targets
Tolerant to crude samples
Can be heated to 90°C for target denaturation, sample lysis
Includes dye for real time fluorescent detection
Can be freeze dried for ambient storage
If you could design the perfect LAMP Master Mix product, which of the following characteristics are
most important? (choose your top 3)
Last Question
ConclusionsLAMP Represents a Powerful Tool for Simplified, Sensitive Nucleic Acid Detection
• LAMP is highly sensitive, specific and rapid
• LAMP assay design tends to be more challenging than PCR due to requirements for multiple (6) long (~40-45 mer) amplification primers
• Achieving robust assay performance requires assay optimization
• Major optimization parameters include assay temperature, enzyme conc., primer design and conc., Mg2+ conc., and additives such as betaine
• Use of DOE may help identify an optimal buffer composition that meets sensitivity and specificity needs
• An optimized LAMP assay could provide point of care/ point of need assay that is economical and easy to implement
ResourcesLAMP Primer Design: PDF explain primer design: http://bit.ly/LAMP-primer-designSoftware PrimerExplorer to design primers: http://bit.ly/2fxAA8ZSoftware LAMP designer to design primers: http://bit.ly/lampdesigner
OmniAmp® Publication:http://bit.ly/omniamp
Questions? www.lucigen.com
Lucigen Tech [email protected](888) 575-9695(608) 831-90118 am – 5 pm Central Time
Thank You!