I am working on SNP through LAMP, but facing few issues on false naked eye detection in positive samples. I have checked and detected SNP through PCR by running F3 and B3 primers and even got bands after LAMP for positive samples, but the problem is after adding HNB (120 uM) positive samples should change violet to sky-blue but color remained same and didn't distinguish between + and -ive samples. How to troubleshoot this issue, kind suggestions are required, please. Photo is attached below.
Hi Muhammad,
I have good hands on experience with LAMP PCR. From the picture I can see too much bands.. try to adjust the annealing temperature. The color change is mainly depend on the pH of the solution. Check the concentration of MgSo4 used in the stock and working solution. For these many number of bands definitely you should have color transformation since the dye does not differentiate specific and non specific amplifications. Hence, theoretically and technically there should be a minor problem in concentration of PCR ingredients only...
Dear Muhammad
This may be false-positive LAMP PCR.Driven by self-priming of generated stem loops, spurious LAMP-like amplification can result from non-specific binding events and produce convincing false-positive signal. I send an article that helpful for you
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