The document discusses the Loop-mediated isothermal amplification (LAMP) technique for detecting DNA. It provides: 1. A brief history of LAMP's development from 1998-2002 and that it has been used to detect over 200 genes/species. 2. Details of typical LAMP reaction parameters, including a temperature of 60-65°C, reaction volume of 25μl, enzymes and primer concentrations used. 3. An overview of LAMP's advantages as a rapid and inexpensive diagnostic technique that works under isothermal conditions, and its disadvantages including more difficult primer design than PCR.

1. LAMP ASSAY Page 1
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)
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, DrPH
Associate Professor IV

2. LAMP ASSAY Page 2
LAMP Reaction Parameters
 Temperature: Isothermal60-65o
C, typically ~63o
C
 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 Applications
LAMP is well-suited for:
1. Limited resource situations
2. Rapid testing
LAMP is not best suited for:
 Detection of unknown or unsequenced targets
Advantages and Disadvantages of LAMP
Advantages
1. Rapid
2. Sensitive and Specific
3. Isothermal
4. Simpler and cheaper equipment
Disadvantages
1. More difficult primer design than PCR
2. Most detection methods are not sequence-
specific
3. Difficult to run multiplex LAMP reactions in single
tube
LAMP Primer Design Steps
 Define the LAMP assay requirements
1. Targets: species/strains that must amplify
2. 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+
Characteristics of LAMP Amplicons
(“Lamplicons”)

3. LAMP ASSAY Page 3
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
 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