History of LAMP 1998: LAMP developed by Eiken Chemical

Company, Tokyo, Japan 2000: first LAMP publication ~2002: Commercial Loopamp™reagent kits

available from Eiken Chemical Company

Demonstrated LAMP targets >200 genes/species have been detected by

LAMP Wide range of targets (selected examples

below)

LAMP ASSAY Page 1

These qualities are presented by the loop-mediated isothermal amplification (LAMP) method.

LOOP MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)[Promising rapid laboratory technique for animal disease diagnosis]

Dr. Clarissa Yvonne J. Domingo, DVM, MPH, DrPHAssociate Professor IV

LAMP Reaction Parameters Temperature: Isothermal 60-65oC, typically

~63oC Reaction volume: Typically 25 µl Enzymes: Bst DNA polymerase, typically 8

U/reaction Primers

Inner Primers (FIP, BIP): Typically 1.6 μM Outer Primers (F3, B3): Typically 0.2 μM Loop Primers (LF, LB): Typically 0.8 μM

dNTP’s: Typically 1.4 mM each Sample: Typically 5 µl/reaction Reaction Time: ~1 hr

LAMP is Best-Suited for Certain ApplicationsLAMP is well-suited for:

1. Limited resource situations2. Rapid testing

LAMP is not best suited for:

Detection of unknown or unsequenced targets

Advantages and Disadvantages of LAMPAdvantages

1. Rapid2. Sensitive and Specific3. Isothermal4. Simpler and cheaper equipment

Disadvantages1. More difficult primer design than PCR2. Most detection methods are not sequence-

specific3. Difficult to run multiplex LAMP reactions in

single tube

LAMP Primer Design Steps Define the LAMP assay requirements

1. Targets: species/strains that must amplify2. Non-targets: species/strains that must not

amplify Gather gene sequences for all available

targets and non-targets Identify conserved gene regions (present in all

target species/strains) Identify gene regions unique to desired targets

(not present in non-target species/strains) Use automated software to identify candidate

LAMP primer sets

LAMP Primer Design: Primer Explorer PrimerExplorer is an automated LAMP

primer design tool provided by Fujitsu Ltd. And Eiken Chemical Company

Available online at http://primerexplorer.jp/e/

Latest Version: V4 Windows operating system, Internet

Explorer 5.0+

LAMP ASSAY Page 2

Characteristics of LAMP Amplicons (“Lamplicons”)

How it is used to detect the presence or absence of DNA

LAMP Detection Method(1) Turbidity

LAMP buffer contains Magnesium sulfate LAMP reaction = DNA amplification DNA polymerization during amplification

produces pyrophosphate By product precipitates as Mg

pyrophosphate Produced in proportional amount to

amplified DNA Visually seen as turbidity which is positive

(2) Sybr green dye test

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An intercalating dye (Sybr Green I) is added to the final LAMP reaction

A visual color change from orange to green in the LAMP product indicates a positive result

(3) Fluorescence test Upon addition of Sybr green dye, presence

of double stranded DNA during amplification increases fluorescence of the cat ion-modulated dye after precipitation of cat ions.

White or greenish fluorescence in the tube when seen under the UV light.

Gel Electrophoresis If an agarose gel electrophoresis of the

LAMP product is performed, bands with various sizes will be visualized at regular intervals.

End of Reaction

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