As the amplification in LAMP is too high, the possibility of carry over contamination is thousand times more then in PCR, there are two remedies for it.

1) stop doing LAMP for some time. change the working place.

2) go through my recent article in JCM

LAMP is a very sensitive method due to the use of six primers instead of two so make 2 - 3 set of primers and try with each. composition of all components is also very important. so make many dilution of each component as primes, buffer, taq, DNA etc. and use each one by one.

I also tried LAMP amplification a couple of times for diagnosis of human african trypanosomiasis and I never got consistent results. Usually appeared false positives, sometimes incongruences in dilutions (serial dilutions of the sample and positive-negative alternatively...). It is why I decided to use other more reliable techniques for me.

Hi Carlos,

I used LAMP method before, indeed, sometimes it is too sensitive! Have you tried to design new primers which would be more specific? According to your case, it seems more complicated than contamination. The system of LAMP is very comprised, with high concentration of salt and 6 primers. Especially the inside primers are very long which may cause secondary structure. Before, design new primers, you can also try to do the amplification without outside primers, and/or repeat the amplification in another lab which has no relevant to your topic and prepare the experiment with their pippets! To see if false positive is still present.

Good luck!

My Primers are very much specific for target. Even we redesign a new set of primers, but results are same, false positive.

false positive result is due to carryover contamination(air contamination of previous pcr product). it is possible to avoid this problem. please refere to following article

http://pubs.rsc.org/en/Content/ArticleLanding/2014/CC/C4CC00540F#!divAbstract

I have encountered the same problem with consistently false positive samples. I have tried different primer sets, titrations of primers and other components of the reaction, tried temperature gradients, kits from different suppliers (Lucigen and NEB), etc. and I am still unable to get rid of the false positive results. Does anyone now what the likelihood of amplification by partial primer binding to random DNA is in an isothermal reaction, considering that according to BALST the primers are highly specific for my target organism/gene (Leishmania donovani/18s rDNA gene). The primers are actually from a publication (Nzelu et al., 2014), which had worked fine for the authors. Is there a trick to this method?

That's the peculiarity with LAMP technology and hence many good researchers do not rely on it. Even if you luckily get a positive result, it more than often, tuns out to be an artifact.

@bhagwan batule can you please provide me the research article which you mention in your comment. Because its not freely available.

Hello,

I'm also adjusting different LAMP reactions with different species and different targets. The problem you mention, false positives in serial dilutions, occurred to me in all cases. It is inevitable and adjust recciones is very complicated. However, I have found better results if I add the loopprimers to the reaction and using the Bst 2.0 WarmStart.

Hi, In my case the most common problem is with marginal contamination of stock primers. I had not problems with establishing LAMP for any bacteria (ex. Salmonella sp., Brucella sp.) but in case of viral DNA or RNA it is extremaly easy to contaminate both primer stocks or reaction solution. For me the change of laminar cabitet worked.

In LAMP, primer concentration play a big role. so i thing you should make new set of primers and try new primers with different concentration of each primer. and use a new Bst DNA polymerase also.

I ordered fresh set of primers from Sigma and used new enzyme also, but getting same false positive results. Negative Controls (No DNA only Autoclave water) also came positive.

As the amplification in LAMP is too high, the possibility of carry over contamination is thousand times more then in PCR, there are two remedies for it.

1) stop doing LAMP for some time. change the working place.

2) go through my recent article in JCM

Yes, this is do to carryover contamination of amplified LAMB product, because LAMB assay produce large copy of product than PCR. You will get contamination while gel running or opening of tubes after reaction. Avoid gel running of LAMP product this will give more contamination. So, please change all reagent for LAMP, including enzyme (stock) and primer (stock) and do again. Please do not open tubes after reaction, lot of methods are published to do LAMB with opening the tube. Better use filter tips and change your pipette.

Many people mention the carryover contamination which is possible if you are not careful with opening tubes. However even if you never open a tube post-reaction there are some LAMP primer sets that are prone to spontaneous amplification, for example by amplification of primer dimers. This can be difficult to predict, but any detection technique such as turbidity, SYBR Green, calcein, hydroxynaphthol blue, etc that relies upon detection of total DNA synthesis can not distinguish between true and false positives. It is preferable if you can adapt a probe-based technique- there are a few out there for LAMP, although not widely adopted, that can reduce the incidence of false-positive detection, although even so it is best to start with a primer set that is inherently less prone to the spontaneous amplification.

How do I eliminate false positives while running a LAMP reaction?

Should make sure to keep pre and post reaction areas separate and aliquots in your most 'clean' place. (positive controls separate too!) To double check primers (esp if trying to multiplex) in silico, should use both: https://www.thermofisher.com/ch/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html and http://biotools.nubic.northwestern.edu/OligoCalc.html It's really about much more than primer dimers! The 'quenched fluorescence' or Quasr method is one concrete was to provide more specificity in detecting the correct amplicons. for instance, see GMO Detective (and now CoronaDetective hopes, with JOGL...) method https://gmodetective.com/science/ (i.e. one primer has FAM tag which is quenched by a short complementary primer with BHQ1 at 3' for instance - after incorp in amplicon, FAM is free to fluor)

Thank you for your reply, Rachel.

Manaswini