Loop-Mediated Isothermal Amplification (LAMP) - Lucigen€¦ · Loop-Mediated Isothermal...

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

0

10

20

30

40

50

60

0.2X 0.4X 0.6X 0.8X 1.0X 1.2X 1.4X 1.6X 1.8X

Aver

age

TTR

(min

)

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

0.00

10.00

20.00

30.00

40.00

50.00

60.00

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

.2

pH 8

.3

pH 8

.4

pH 8

.5

pH 8

.6

pH 8

.7

pH 8

.8

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

0

10

20

30

40

50

60

High Medium Low NTC

Tim

e to

Res

ult (

min

)

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 Supporttechsupport@lucigen.com(888) 575-9695(608) 831-90118 am – 5 pm Central Time

Thank You!