Hi Javier,

Following is the reagent I used in my LAMP reaction:

1.FIP & BIP

2. F3 & B3

3. dNTPs

4. Bst Polymerase (large fragment)

5. MgCl2

6. Betaine

7. Buffer

8. H2O

There is no order in addition of reagents. I make the LAMP master mix in ice and add the Bst polymerase at the end. Make sure you add same amount of template DNA to get consistent result. Aliquot all your LAMP reagent in small volumes and store it at -20. Repeated freeze thawing can reduce the efficiency of enzyme.

Did you get false positive result? If yes, one of the possible reason for your problem is contamination. Amplified LAMP product is very volatile and easy to get contaminated.

Some of the precautions I take to minimize contamination:

1. Use separate pipettors to mix LAMP reagents and load amplified product into agarose gel

2. Don't open the tube (after reaction) inside the laminar flow hood/ bio safety cabinet .

3. Use separate working place to make LAMP master mix and loading amplified LAMP product into gel.

See link below for better understanding.

http://www.nature.com/nprot/journal/v3/n5/full/nprot.2008.57.html

strangely as it may seem in my experience the order of adding the reagents did make a difference. Buffer, MgCl2, dNTPs, primers (mixed together as a working concentration), water and the Bst polymerase with the DNA template last. Most importantly we don't open tumes that contain LAMP products at all. The results are analysed by visual inspection of the tube under UV light to see the turbidity if you don't have access to a turbidimeter that reads the results in real-time.

I hope this helps.

Javier,

I am struggling with the same issues with my studies. I believe I have tried everything under the sun to obtain reproducible data but, without success, continue to require changes to my methods.

I'll keep your post in mind if/when I have a breakthrough.

Mike

Hello everyone;

Finally I managed to successfully to reproduce the results. Bst polymerase 2.0 WS is the best results has given. Also, I think it is important the order of addition of the reagents:

Water + Betaine + MgSO4 + Buffer + dNTPs + primer + Bst DNA

I hope this helps

Regards

Hi all,

I am currently conducting a research on the primer design as many LAMP users find it difficult to get their way around getting a validated and optimised functional primer.

I was wondering if their are any assay that are any assay that are optimised and validated ready to use assays/primers for specific target. would it not safe time and add to the convenience besides getting a functional primer ?....

Javier I am having the same problem that you had, did that order of adding the reagents help you acquire the most optimal results?

I used Combination of MgSO4 and primers as one reagent and remainng reagent togetheras another reagent. The two separate reagent are quite stable for months at -20. I never such difficultes.

I am working with miRNAs and I ca not find a kit to perform a LAMP reaction; so, I am preparing the LAMP reaction without the Loopamp kit, with the components separately. At first it was working but now the reaction became slow.  I replaced whole reagents (except betaine and DNTps) but I could not find any result.  

Hi karen, how do you have the primers? diluted in the fridge?

Hi Marta. I have them diluted (water) at -20.

Hi karen, it means that every time you need to perform a lamp reaction  you use one of the dilutions stored at -20ºC?

Try to do a new dilution from the mother solution and use that one,

Hi! Has anybody notice any difference when using MgCl2 instead of MgSO4 in the mastermix?? Seems my MgSO4 is contaminated and i was thinking that i can use MgCl2 while i wait for a new one to arrive...

Thanks in advance! :)

Hello Andrea! I do not have long experience working with MgCl2 but many people talk me there is not real diference between both reagments. The important ion is the Mg 2+. However all labs recomend MgSO4. I think you should try, each reaction is diferent.

Good luck

 Gracias Javier!

I'll try it while i wait for the MgSO4 ...hope it works! 

hi

Try this enzyme with an optimized buffer:

https://www.protean.bio/en/recombinant-protein/124/bsti-dna-polymerase

Hi All!!

I have started working on LAMP recently with Bst polymerase large fragment(NEB). I have designed two sets of primers for two different region. One set is working fine but for another target not. my other target is AT rich. Can anyone tell me how amplify AT rich region in LAMP.

Thanks

Hi Swati, if you are working with an AT rich region you need to pay attention to betaine concentration. If the target region has a GC content lower than 45%, addition of betaine zome times is not needed. Make a titration by lowering the betaine concentration from your current working conditions till no addition of it to reactions. Maybe it can help.

hi everyone

i am working in RT-LAMP with WarmStart® LAMP Kit (DNA & RNA), and this kit gives results that can be read by real time pcr instruments as it has a dye act like sybergreen. i have made a validation for the kit and primers including loop primers and both worked perfectly for 3 runs, then it began to give strong false positive with negative samples and controls. i do not understand what is wrong with the kits, could i find any help in explanation of this misreading?

Hi all and Javier,

I got the LAMP reaction with a good amplification and I could see the change of color (HNB), this worked very well for two week and then I didn't have more amplification. I tested all reagents separately and works fine, so I don't know what happend, I would like to know if you could solve your problem and help me with any suggestion about that.

Best

Angela

Hello Angela Carrasco . Did you check the fidelity of your DNA to confirm if it is still intact and not degraded already? Also, kindly post an update here if you were able to get around the problem. Thank you.

Hi Amany Adel, I was having the same problems in the past. I was having an unespecific amplification in my no-template controls. I could temporarily solve it by aliquoting primers in small volumes to make one-use aliquots. In the end, I had to design new primers for the same region and I did not experience these problems anymore.

Otavio Espindola

thanks a lot for your response

i have tried anther trend in dealing with this problem and i think the results were satisfied

i had added DMSO with final conc. 5% and heating at 67c , the non specific reaction was disappeared

Hello Godwin Attah Obande , thanks so much for your suggestion, and yes I tested all the reactive even the quality of the DNA (doing PCR with different specific primer for my sample).

So I tried other things, I changed the dye because some time the result was difficult to observe well, therefor I had to adjust a new protocol but using the same primer, dNTPs, DNA, buffer and water. I have the same situation that last time, finally I had very good results but only in the beginning because when I did the repetition I had cross contamination and the blanck tourn the color.

So I don't know which is the problem!!! I changed all reactives, I used a new stock of everything, I am very careful when I go to work in the laboratory, I clean the space that I use very well, I leave the material with which I am going to work in UV, I keep the reactives in ice. But my big problem rigth now is that al samples change color, the positive samples, negative samples and controls.

Please if someone has gone through a similar situation please help me with any comments or tips

Hi @Angela Carrasco , were you able to find a solution..did you add DMSO to the reaction?

Hi everyone,

I am doing an RT-LAMP technique for RNA Tembusu virus detection. I used pre-adding HNB for the direct detection by the naked eye. In the beginning, the RT-LAMP reactions seem to work well. Then it began to give strong false positives with negative samples and controls. I did not open the tubes after amplification. I checked the contamination by RT-PCR but it's negative. Then I reduced the amplification time to avoid non-specific amplification. However, the results are still not stable. Even, I mixed new reagents for RT-LAMP and I tested with another RNA template, it works well. But, for the RNA Tembusu virus, sometimes negative control is negative and some are positive.

Please help me with some suggestions!

Hello Ngan Thi Mai. Have you checked to be sure that your LAMP primer set does not generate dimers or hair pins? That could usually cause false positive results in the absence of contamination.

Godwin Attah Obande Thank you very much for your suggestion. Ys, I did and no results of generating dimers as well as hairpins of primers.

There is no ladder like pattern but two or three bands were obtained near the wells along with primer dimer at the base in the positive control. Earlier, the results were clear cut. I have changed all the reagent, amplification conditions, volume of template DNA, etc. though nothing helped me out. Pl suggest what should i need to change

Ngan Thi Mai：Hi, Have you used high concentration positive control (plasmids or whatever) in your environment? Use of such positive control may lead to false positive results

I am currently working in LAMP. It's really frustrating. I have been using 2x warmmstart LAMP colorimetric reagent. I have designed two set of primers. One was working good and another does not. Everything was going good till I optimized the reaction temperature and time. But for the specificity, the results are not good. I am having false positive even in the negative samples. However, my NTC is negative.

What could be the possible solutions for this problem? Please suggest me.

Thank you.

Deepa Chaudhary

[email protected]

Deepa Chaudhary carry over contamination of amplified LAMB products is quite common so try to replace all your reagents, change pipette, use filter tips and never open a tube post reaction.Avoid gel running of LAMP products and detect amplification using a technique such as calcein, hydroxynaphthol blue, SYBR Green, turbidity etc. Hope this helps. E

Negatives being positives can be due to primer dimer formation. Have you tried using Betaine or DMSO to avoid such primer dimer formation and thus giving false positives?

I'm starting work with lamp trying to reproduce a previously standardized protololo. I bought everything new (enzyme primers) however the banding pattern of my control (reaction without DNA) is identical to the reaction containing template. Has anyone ever experienced this?

Eleni Makraki Thank you so much. I replaced all the old reagents, primers, clean the pipette. It worked.

Bushran N Iqbal Thank you for the response.

Luana Parras Meleiro yes I have experienced the hardest time with LAMP assay reaction due to the false positive. LAMP method is very sensitive. It has a very high chance of getting contamination and resulting in false positives.

In my case, I have changed all the old reagents, primers, pipettes. We found root cause of our contamination was my primer and reagents were contaminated. The LAMP reaction should be handled properly. If you want to verify the LAMP products results with gel electrophoresis, you should have a separate place for running gel rather than the LAMP reaction preparation areas.

Luana Parras Meleiro Another probable reason might be due to primer dimer. If you don't have specific primers, then it can cause primer dimers resulting in false positives. Check your primer as well. Thank you.

@Deepa Chaushary I'm probably having problems with dimmer primers. I've used betaine and it didn't work. do you have any suggestions for this problem?

Deepa Chaudhary you're welcome!

Luana Parras Meleiro try DMSO

Deepa Chaudhary, Has your problem been definitively resolved?

Luana Parras Meleiro Yes, Mine was the case with contamination. So when I changed everything including DNA, the problem was solved. But in your case, as you mentioned even if you used betaine, it did not work. So I suspect the primer itself. Designing a suitable primer is very important for LAMP assay. How did you design the primer? Did you compare your primers to the multiple sequence alignment for the corresponding target locus?