LAMP[LOOP MEDIATED

ISOTHERMAL AMPLIFICATION]

Dr. Arifa Akram BarnaMBBS MD (Virology)IEDCR, DGHS, Dhaka

INTRODUCTION:

Nucleic acid amplification is a valuable tool for the diagnosis of infectious diseases.

Several amplification methods are available including PCR, Nucleic acid sequence based amplification (NASBA), Self sustained sequence replication (3SR) and Strand displacement amplification (SDA). Among these, PCR is the most widely used in various forms such as reverse transcription PCR (RT-PCR), nested PCR and multiplex PCR.

Criterion for ideal diagnostic tests:

The criterion for ideal diagnostic tests (According to World Health Organization) are

(1)Sensitivity, (2) Specificity ,(3) Considerably of low cost,(4) Simplicity ,(5) Rapidity (6) Adoptable to any sort of climatic

variation and (7) Easy availability of instruments .

•LAMP is a powerful innovative gene amplification technique emerging as a simple rapid diagnostic tool for early detection and identification of microbial diseases .

•The whole procedure is very simple and rapid which in the amplification can be completed in less than 1 hour under isothermal conditions by incubating the mixture of samples, primers, Bst polymerase with strand displacement activity and substrates all the in a single tube.

•LAMP provides high amplification efficiency, with DNA being amplified 109–1010 copies in 30–60 min.

What is LAMP?

• Because the efficacy of PCR amplification may be affected by the presence of a number of inhibitors of Taq polymerase enzyme in the serum such as EDTA, phenol, polyamines, polysaccharides and calcium alginate, DNA extraction is required before amplification .

• It has been shown that LAMP exhibits less sensitivity to inhibitory substances present in biological samples than PCR. This robustness of LAMP against inhibitors can contribute to saving the time and cost required for sample processing steps.

LAMP method has the advantage of amplifying the target DNA from partially processed and/or non-processed sample .This exclusion of DNA extraction step not only shortens the reaction time and result interpretation but also eliminates the chance of contamination .Bst DNA polymerase with strand displacement activity at 65℃ The whole amplification to 109 – 1010 copies is finished within 15 to 60 min at 65℃, isothermallyAmplification 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

Properties

PCR LAMP

Denaturation Requires for separation of strand Not mandatory as enzyme displace strand

Primers Require 2 primer-Forward & Reverse.

Require 6 primer

Annealing, Extension

Usually employs 3 steps, working at different temperature & timing.

Works under constant temp which should, usually between 60-65˚c

Time Required Takes 2-3 hr 60 min usually.

Post amplification process

Needs agarose gel electrophoresis for knowing the result.

By incorporating dye like SYBR Green or naked eye.

Sensitivity Can detect up to ng level of DNA Can detect up to fg level of DNA

Instruments Needs sophisticated instruments in order to maintain different temp within a given time.

No need of expensive instruments but only water bath or Heat block.

Require template preparation

Requires which should be pure & impurities can hinder PCR reaction.

No need for processing of DNA.

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Design of LAMP primers 4 types of primers based on the 6 distinct regions of the target gene: the F3c, F2c and F1c regions at the 3' side and the B1, B2 and B3 regions at the 5' side

FIP Forward Inner Primer (FIP) consists of the F2 region (at the 3' end) that is complementary to the F2c region, and the same sequence as the F1c region at the 5' end

F3 Primer

Forward Outer Primer consists of the F3 region that is complementary to the F3c region

BIP Backward Inner Primer (BIP) consists of the B2 region (at the 3' end) that is complementary to the B2c region, and the same sequence as the B1c region at the 5' end

B3 Primer

Backward Outer Primer consists of the B3 region that is complementary to the B3c region

LF/LB Two loop primers - forward loop primer (FLP) and backward loop primer (BLP) accelerate the amplification reaction by binding to additional sites that are not accessed by internal primers

PRINCIPLE OF LAMP

(A) Non-Cyclic Steps : generation of stem loop DNA with dumbbell-shaped structure at both ends

One of the LAMP primers anneal to the complimentary sequence of double stranded target DNA Initiates DNA synthesis using the DNA polymerase with strand displacement activity, displacing and releasing a single stranded DNAUnlike PCR, no need for heat denaturation of ds DNA

STEP 1

Through the activity of DNA polymerase with strand displacement activity, a DNA strand complementary to the template DNA is synthesized, starting from the 3' end of the F2 region of the FIP

STEP 2

The F3 Primer anneals to the F3c region, outside of FIP, on the target DNA and Initiates strand displacement DNA synthesis, releasing the FIP-linked complementary strand

STEP 3

A double strand is formed from the DNA strand synthesized from the F3 Primer and the template DNA strand

STEP 4

The FIP-linked complementary strand is released as a single strand because of the displacement by the DNA strand synthesized from the F3 PrimerReleased single strand forms a stem-loop structure at the 5' end because of the complementary F1c and F1 regions

STEP 5

This single strand DNA in Step (5) serves as a template for BIP-initiated DNA synthesis and subsequent B3-primed strand displacement DNA synthesis

STEP 6

Double stranded DNA is produced through the processes described in Step (6)

STEP 7

The BIP-linked complementary strand displaced in Step (6) forms a structure with stem-loops at each end, which looks like a dumbbell structureThis structure serves as the starting structure for the amplification cycle in the LAMP method (LAMP cycling)

STEP 8

(A) Non-Cyclic Step : generation of stem loop DNA with dumbbell-shaped structure at both ends that is ready to enter into cyclic amplification step

(B) Cycling amplification step

B) Cycling amplification step After forming a dumbbell-like structure,

the amplification cycle in the LAMP method begins. At this step, Loop Primer Forward (LPF) and Loop Primer Backward (LPB) bind with their complementary sequences (F2c and B2 region respectively) and primed the amplification. At each cycle of amplification, the number and the size of the existing products increased. Thus, at the end of the reaction the products can be resolved by agarose gel electrophoresis as a ladder like band instead of single band

Using Loop Primers

Time saving by Loop Primer……

Time required for amplification with Loop Primers is one-third to one-half of that without Loop PrimerWith the use of Loop Primers, amplification can be achieved within 30 minutes

Original method: no Loop PrimerRapid method: with Loop Primers

LAMP does not require thermal cycler

Cycle reaction Denature (95℃) Annealing (50~60℃) Polymerization (72℃) ~ >1 hour

• Isothermal reaction

• 60- 65℃• Max 1 hour

PCR LAMP

Thermopol buffer (10x) 2.5 µl

MgSO4 (50 mM/µl) 4.0 µl

dNTP mix (10 mM/µl) 3.5 µl

F3 Primer (10 µM/µl) 0.5 µl

B3 primer (10 µM/µl) 0.5 µl

FIP Primer (100 µM/µl) 0.5 µl

BIP primer (100 µM/µl) 0.5 µl

F-Loop primer (100 µM/µl) 0.25 µl

B-Loop primer (100 µM/µl) 0.25 µl

Betaine (5M) 5.0 µl

Bst Polymerase 1.0 µl

Water 5.5 µl

Template 1.0 µl

Total 25.0 µl

Procedure

LAMP Reagents

•Bst Polymerase:

The LAMP polymerase enzyme which plays a very crucial role, it can be considered as the “brain box” of the technique. Bst DNA Polymerase, Large Fragment is the portion of the Bacillus stearothermophilus DNA Polymerase protein that contains the 5´→3´ polymerase activity, but lacks 5´→3´ exonuclease activity. Bst DNA polymerase does exhibit greater tolerance to inhibitors typically found in diagnostic specimen (e,g blood, humic acid), which is an advantage as compared to PCR polymerase. The optimal reaction temperature for Bst polymerase is between 55-65°C (107). At temperatures above 70°C, enzyme activity is significantly decreased while at 80°C the enzyme is inactivated.

•Magnesium sulphate Magnesium sulphate which forms magnesium pyrophosphate during the course of the reaction which enables to visualize the result based on the turbidity formed.

•Betaine Betaine was used in the LAMP reaction mixture to reduce base stacking and to increase not only the overall rate of reaction but also target selectivity by significantly reducing amplification of irrelevant sequences. It also destabilize the DNA helix and negates the need for heat denaturation of the template. It is theorized that reaction temperatures of 65°C in conjunction with betaine are sufficient to facilitate strand separation; however, the exact mechanism is unknown

Detection

Visually1.Fluorescence – Eye2.Turbidity – Turbidimeter (or Eye)

Gel electrophoresisFluorescence color changeELISAICT

Detection methods Visual methods

Real time

Optigene instrument for LAMP

LAMP product is visible to naked eyes

(DNA)n-1+dNTP → (DNA)n + P2O74-

P2O74- + 2Mg2+ → Mg2P2O7 ↓ PPT

Insoluble Turbid

Detection using a fluorescent metal indicator (calcein)

Green fluorescence

by free calcein

Loopamp Real time Turbidimeter

With the time and temperature preset, gene amplification will occur, and the detection can be done by simultaneously monitoring the white turbidity caused by the existence of magnesium pyrophosphate, the amplification by-products

LAMP Reagents in Dried Form

Dried LAMP reagents fixed on the lids

↓ Reconstitute by PURE

treated sample solutions

Save steps for preparing LAMP reaction solutionsStable at room temperature for at least one year （ no need to store in the fridge ）

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