(LAMP) Problems with Loop-mediated isothermal amplification

[acnaria]

By the way we are using DMSO instead of Betaine do you think DMS could be causing my false positives or in some way it's activating my calcein???

[jensf]

Have you tested your calcein results with other detection methods? My samples are always green (+) when using calcein and gel also shows false positive results. The detection system with calcein and manganese-ions is working well.

When preparing mastermix, the last you should add is the manganese. Then you should see whether DMSO is inactivating the calcein. I'm using betaine.

[jensf]

I also tried DMSO instead of Betaine. Results are equal! Has someone new ideas for the false-positive problem?

[Babak]

Hi ,


I am faced with almost same problem of non specific amplification. I worked with two different primers set. for almost a month& half, everything was working well and then it start to have non specific band in my negative control. I renewed all the reagents but it seems that I have the same problem. does anyone faced with the same problem? if so, do you have any solution for it?


Thanks
Babak

[msarah]

Has anyone found the reason for the false positive results

[mcalapai]

I was also having trouble with false positives. I changed my MgSO4 concentration (from 7mM to around 10mM) and it initially worked for a little bit (a month or so). After that I kept getting false positives again.

Then we decided to mix and match primer sets (on a whim and because nothing else was working) and found that using the same primer set for BIP and FIP and using different primer set for F3 and B3 respectively worked with no amplification in the negative. repeatedly!

I had a total of 3 primer sets (FIP,BIP, F3,B3 in a set) to work with.

So currently I am using FIP and BIP from one primer set. F3 from another, and B3 from another to amplify the same gene.


I will let you know how it works out but for the past 2 weeks its been working great.

[jensf]

Hi,

it's still the same problem. When running LAMP without dNTP, no reaction takes place. Normal LAMP results in false positive in NTC. I also changed concentrations of betaine, mgso and results are the same. I made all buffers new...still same.

So i think the primers are contaminated somehow or the primers are hybridizing and amplifing. But in case of self-annealing and amplification, is it possible to get ladder-like band pattern on gel?

Maybe the long primers (FIP/ BIP) are very sensible because they are much longer than normal.

Primer redesign never solved problem. In case of contamination, how to get rid of it? Room separation for Mastermix, reaction and detection is done?!

Originally Posted by msarah

Have you solved your problem yet. We are running two different primer sets for completely different target genes. One consistently gives us good results and the other has false positive. The Bst pol is the same for both runs. I am beginning to think that the primers might be reacting with each other in some way. We use the lower concentration of Betaine 0.8 M which increases the stringency. The next step in our plan is the move the primers slightly and check very carefully that there is absolutely no change that they are annealing to each other and giving the false positive. As far as Acinaria having + results with no primer present is very strange. We had positive results when no DNA template was present , but - results when BST or dNTPs were not present. this leads me to believe that the primers are reacting with each other and have to be replanned.

[jensf]

Exactly the same...any ideas!?????

Originally Posted by Babak

Hi ,


I am faced with almost same problem of non specific amplification. I worked with two different primers set. for almost a month& half, everything was working well and then it start to have non specific band in my negative control. I renewed all the reagents but it seems that I have the same problem. does anyone faced with the same problem? if so, do you have any solution for it?


Thanks
Babak

[Babak]

I faced with the NSA problem. In this case I designed the new sets of primers and it worked properly. So I suggest anyone that face with this problem:
1. change all sets of reagent. If still you have the problem don't use those primers anymore . instead, design new sets of primers and work with them.

Best,
Babak

[Babak]

Anyone knows how to design primers for LAMP?

Thanks
Babak