Can the reason be a different Salmonella strain? Try shorter incubation/lower primer cc in negative controls and see whether that helps

These results track with typical LAMP issues; it is very easy for contamination to occur, much more so than any other form of DNA amplification due to the mechanism of the amplification forming non-specific structures. Because the loop primers are not fully necessary, it also makes sense that not using these would lead to fewer false positives. There are several steps that you can take to minimize false positive reactions, such as (1) truncating your FIP primer by 2 base pairs on the 3' end to avoid 'LIMA' or 'UIMA' mechanisms for nonspecific amplification from the primers (2) checking primers for potential dimers or self-dimers, and (3) experimenting with the incubation temperature and time to determine if there is a time and temperature in which only true positive amplification occurs.

However, the best advice I could give would be to NEVER work in the same area with the same materials pre-amplification and post-amplification. Contamination is so easy in LAMP. Try using a new primer stock and a new LAMP mix and working in a completely new area to set up the amplifications.

Tom Kasputis Hi!

I’m currently facing the same issue as the original poster — I’ve also observed occasional amplification in my NTCs, and I suspect it may be due to the LIMA or UIMA mechanisms you described.

I saw your suggestion to truncate the FIP primer by 2 base pairs at the 3′ end to help mitigate this, and I’d really appreciate it if you could share more details on how exactly you perform the truncation. For example, do you remove the 2 bases directly from the F1c region, or from the end of the full FIP sequence (F1c–TTTT–F2)?

I’ve also been trying other strategies in parallel, such as lowering primer concentrations and adding DMSO, but I’m very interested in trying your approach as well.

Thank you so much in advance!

Best regards, Hayden