The world's scientific and social network for malaria professionals
9425 malaria professionals are enjoying the free benefits of MalariaWorld today

Loop-Mediated Isothermal Amplification Method (LAMP): Low and Effective cost Novel Tool for Molecular Public Health

December 9, 2009 - 05:35 -- Usa Lek-Uthai

This technique need not to have even high cost equipments (especially PCR or GelDoc or refrigerator). The specific LAMP primer set consist of F3(forward outer primer), B3 (backward outer primer), FIP (forward inner primer), and BIP (backward inner primer) These primers will be designed using the Primer Explorer program (http://www.loopamp.eiken.co.jp) to amplify the 18S ribosomal
RNA gene (http://www.ebi.ac.uk.com). The protocol could be optimized for each reaction; my experience for the study of Acanthamoeba spp rapid identification, LAMP was performed for 60 min at temp 65 C in 25 microL of a mixture containing 2 microL of extracted DNA, 40 pmol each of FIP and BIP, 5 pmol each of F3 and B3, 1.4 mM deoxynucleoside triphosphates, 0.8 M betaine and 1 microL of Bst DNA polymerase in buffer (20 mM Tris–HCl pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, and 0.1% Tween 20). The reaction was terminated by heating (using water bath or heating block) for 2 min at temp 80 C. Turbidity of the LAMP reaction will be inspected visually in real time. The DNA products could also be visualized under UV light after addition of Loopamp fluorescent detection reagent (Eiken Chemical Co., Ltd.). LAMP products was confirmed by electrophoresis in 1.5% agarose gel and visualization by staining assay. The sensitivity and specificity were 100%. (see more at: Usa Lek-Uthai et al. Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method. Experimental Parasitology Volume 121, Issue 4, April 2009, Pages 342-345). However, the study of genotyping and copy numbers for the Plasmodium spp specific genes (the species identification need not to have any DNA extraction method from fresh or dried blood spots) in different malaria endemic regions in Thailand since 2007 have been doing well (unpublished). Therefore, this technique might be useful for molecular public health in the developing country’s disease control program. LAMP took less time than qPCR, which required 2 h as compared to 1 h for the LAMP procedure. However, the cost of this technique is the cheapest (about 70 cents US per sample compared to 5–7 $US per sample using DNA amplification by realtime PCR.

Comments

Submitted by Eman (not verified) on

Dear USA

Greetings,

I did not understand the LAMP technique well, would you please write me the procedures? Can I use LAMP test to detect BCR/ABL fusion gene? if yes what is the method?

Thank you

Eman Abbass

Submitted by Francisca (not verified) on

Hi USA,

I have had trouble with negative control (no template)for a few month like others, at first was all OK with LAMP, but suddenly it wasn't work.

I have changed primer stock, betaine, dntp's, MgSO4, buffer, water (I use DNAse free). The mix is made in a different room to that I add the templated (with different pipet, too) I use filter tips.

To probe if I had primers contamination, I did a PCR with: just B3/F3, FIP/BIP and LB/FB primers and one mix with all primers (using Taq and its solutions, of course)with its negative control, respectively.. I realized all negative controls for each reaction were OK. Even the all primer mixed control was OK.
So I repeat the LAMP and again the same result the negative control is a lader pattern-like...will it be possible some LAMP reactive is contaminated?, but I change all a lot of time!..

And in this wall I have read that this problem seems to be comun.

Someone could fix it?

I'll be thankful if one of you could help me!

Submitted by Pratiksha Raghu... (not verified) on

Hi,
We are working on LAMP based diagnostics. We are getting good results with no false positives.Amplification product on agarose gel is always in coordination with HNB based color change.The problem is we observing a smear in positive samples but not getting the typical banding pattern. Is this a problem?, or what can be done to avoid this.
Plz help
Thank u

Submitted by Indra (not verified) on

Hello! Usa
Our laboratory hust started to planning do LAMP. And I am rookie researcher. :D I searched google bout LAMP procedure and required equipments and reaction solutions that are neccesary for LAMP. But i feel like there is something i missed. If its possible can u e-mail me information, LAMP procedure and some equipment list or something else for free? :) Thank u!

Submitted by Uzma (not verified) on

Hello I am trying to perform LAMP for the identification of plant fungal pathogen. Used Bst pol 2 protocol with and without betaine at 65C but did not get any product. I used 4 different sets of primers. However, when I used F3 and B3 primer got amplification of target gene by PCR (annealing was at 54C) by all the four sets of primers. Any suggestion to develop LAMP in this case?

Submitted by Shaona (not verified) on

Dear Uzma,
Hi, I am facing the same problem with my LAMP. F3 B3 primers work in PCR at parasite DNA loads as low as 100copies/ul (55deg annealing) but when I use the same amount of parasite DNA in LAMP I see no amplification. Did you ever figure out the solution to your problem?
I have titrated for primer conc, Mg conc, Bst large fragment/2.0/3.0, temperature (60deg to 68deg), time (30m to 100m) as well as denaturation temp (80deg-2m; 95deg-2m) but nothing seems to work. Any help will be deeply appreciated!

Pages