I am currently working on a project which uses LAMP to detect Bacillus cereus. The specificity test we did earlier has no problem with the No-Template Control, however when we move on to the sensitivity test the NTC starts to turn orange/yellow usually after 50min. The NTC did not change colour after 1 hour when we first test it. Is there any reason why the NTC vessel turn yellow or are there any possible ways to improve the accuracy?
Operationally - never open a tube from a LAMP reaction anywhere close to where you set up a reaction. If you're doing color-change detection, that is a closed-tube technique. That's good. If you consistently never open a tube, then there is no chance of spreading amplicon contamination. If you do open a tube, e.g. to run a gel, do it far away. If you've already done it, then the lab may be contaminated and consider it a lesson learned and shelve that particular assay.
Practically - some LAMP primers more than others are prone to sporadically generate garbage. Depends on the primer set. It may not happen every time. Usually you see it at >1 hour incubation but it can happen faster. It is difficult to predict, but may involve some primer-primer self-amplification, even with things that don't look like perfect matches. There are rumored to be ways to suppress this (additives and such that destabilize imperfect matches or short regions of complementarity) but I have not had a chance to try anything.
Maybe you have solved the problem. Here are some of my suggestions. You should reduce the chance of aerosol pollution in every experiment.
Tips: 1. Strict division of laboratory( Solution preparation area; Sample processing area; Testing area ) 2. Basic Operation( In accordance with the first negative, positive, from low to high concentration gradient, finally added template )3. The operating environment( UV irradiation >500bp; Treatment with DNA degradation reagents ) I hope it worked for you!
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