loop mediated isothermal amplification

1. LAMP
Loop-Mediated Isothermal Amplification
Prof. Abdelraouf A. Elmanama
Islamic University of Gaza

2. WHO recommends that an ideal diagnostic test suitable for
developing, underdeveloped and undeveloped countries
should be:
• Affordable
• Sensitive
• Specific
• User-friendly
• Robust and rapid
• Equipment free
• Deliverable to the end user
“ASSURED”

3. Examples of Molecular methods
• Polymerase chain reaction (PCR)
• Ligase chain reaction (LCR)
• Nucleic acid sequence-based amplification (NASBA)
• Strand Displacement Amplification (SDA)
• FISH (fluorescent in situ hybridization)

4. PCR features
• Easy to develop new assays
• Easy to perform
• Rapid
• Sensitive
• Specific
• Affordable
• Could be used to quantification (Real time)

5. Major Disadvantage
• Thermal cycling
• Denaturing
• Annealing
• Extension
This requires
• A thermal cycler
• Time to complete the cycle
• Programming or reprogramming
of the Thermal cycler

6. LAMP
•Loop-Mediated Isothermal Amplification

7. First publication on LAMP

8. Bst DNA polymerase
Bacillus stearothermophilus DNA Polymerase
• Is with strand displacement activity at 65℃
• The whole amplification to 109 -1010 copies is achieved within 15 to
60 min at 65℃, isothermally.
• Amplification and detection of gene can be completed in a single step
• No need for a step to denature double stranded into a single stranded
form

9. SpecificSimple
Features Rapid
Cost
effective
Isothermal
LAMP

10. Advantages
• The amplification efficiency is extremely high
• Reduced total cost- not require special reagents or sophisticated
equipment.
• Amplification can be done with RNA templates following the same
procedure as with DNA templates, simply through the addition of
reverse transcriptase (RT-LAMP)

12. LAMPPCR
Isothermal (single temperature)Require temperature cycling
Requires 6 primersRequire 2 primers
Rapid (typically <30 minutes)Slow (typically >one hour)
Typical yield ~ 10–20 µgTypical yield ~ 0.2 µg
Amenable to visual detection based on
turbidity etc.
Not amenable to visual detection
Tolerant to sample matrix inhibitorsSensitive to sample matrix inhibitors
Difficult to multiplexCan be multiplexed
PCR Vs. LAMP
Assays are Faster, Simpler Once Developed

13. LAMP Amplification Detection Methods Multiple
Choices Are Available Depending on Needs
• Mg Precipitation
• Colorimetric
• Agarose Gel
• Real Time Turbidity

14. Mg Precipitation
. (A) The turbidity indicating a positive reaction (+) was caused by the magnesium
pyrophosphate precipitate produced during DNA synthesis, whereas the negative control (−)
remained clear. (B) The solution turned green (SYBR Green) (+) in the presence of a LAMP
amplification, while it remained orange (−) with no amplification.
Visual assessment of DNA amplification by LAMP
magnesium pyrophosphate precipitate SYBR Green

15. Metal indicator hydroxynaphtol blue (HBN)
Scheme of color change of HNB after positive
LAMP assay.
Magnesium ions react with pyrophosphate
derived from dNTPs during LAMP reaction
and generate a precipitate, the resulting
depletion of magnesium ions in solution
results in a color change from violet to blue,
indicating a positive reaction

16. Turbidity

17. Goals for Optimization
• Faster Time to Results (TTR) and Lower Background Signals
• Increased reaction speed (faster TTR)
• Decreased non-specific amplification (slower TTR from Negative
Control)
• Improved sensitivity (Detect low copy inputs)

18. Factors for Optimization
Multiple Parameters Should be Assessed
• Primers (design, concentration and ratio)
• Enzyme (concentration)
• Reaction temperature
• Mg ion concentration
• Reaction pH
• Additives such as betaine (amplification enhancer in GC rich nucleic
acid)

19. Applications
• LAMP has the potential to be used as a simple screening assay in the field or at
the point of care by clinicians.
• Because LAMP is isothermal, which eradicates the need for expensive
thermocyclers used in conventional PCR, it may be a particularly useful method
for infectious disease diagnosis in low and middle income countries.
• LAMP is widely being studied for detecting infectious diseases such as
1. Tuberculosis,
2. Malaria,
3. Sleeping sickness and
4. SARS-COV-2.
• In many regions, it has yet to be extensively validated for other common
pathogens.

20. • LAMP has been observed to be less sensitive (more resistant) than
PCR to inhibitors in complex samples such as blood, likely due to use
of a different DNA polymerase (typically Bst – Bacillus
stearothermophilus – DNA polymerase rather than Taq polymerase as
in PCR).
• This feature of LAMP may be useful in low-resource or field settings
where a conventional DNA or RNA extraction prior to diagnostic
testing may be impractical.

21. LAVA: An Open-Source Approach To Designing LAMP
(Loop-Mediated Isothermal Amplification) DNA
Signatures
We have designed and
demonstrated new
software for
identifying signature
candidates
appropriate for
LAMP assays.

22. Summary
• LAMP is highly sensitive and specific DNA/RNA amplification method
• Advantage of LAMP is isothermal reaction condition, hereby LAMP is
affordable because of no need to have expensive thermal cycler.
• Although recommended reagent storage temperature is 20 o C,
reagents can be stored at ambient temperature for at least 2 weeks.
Hereby there is no need to have cold chain for reagent distribution
• Crude DNA preparation can be used as LAMP template DNA.
• Cost of LAMP can be reduced to approximately 1 USD/test or cheaper
• LAMP can be a field molecular diagnostic method for infectious
diseases……….food inspection………environmental testing……….. Etc.

23. ‫شكرا‬‫استماعكم‬‫حلسن‬