Dear Prof. Adolfo Cruz

In your case, this discrepancy indicates that a reaction has occurred (seen by the HNB color change), but it is not the specific, robust amplification needed to produce the characteristic ladder pattern on a gel. During a LAMP reaction, specific primers produce a series of double-stranded DNA amplicons of varying sizes, resulting in a distinct "ladder-like" pattern when separated by gel electrophoresis.

The results so obtained may be the non-specific amplification which is the most common reason for a positive HNB result without a gel ladder. In a LAMP assay, non-specific DNA synthesis can occur due to primer-dimer formation or the primers binding to non-target sequences.

It is also possible that there could be a small amount of DNA contamination in your reagents which can produce a weak or non-specific amplification, resulting in a color change but no observable gel bands. LAMP is extremely sensitive and prone to carryover contamination.

Finally, if it is none of the above, then the problem could be with the gel or the electrophoresis process itself can cause a lack of visible bands. There may be issues like the agarose gel concentration or running buffer that can lead to poor or failed DNA migration. Also, over-running the gel could cause the DNA to migrate off the end of the gel.

You may solve this problem by following the below steps. Maybe it will work! A. Include a no-template control (NTC) with your next set of reactions. If the NTC turns blue, it confirms carryover contamination. To get rid of contamination, use filtered pipette tips, clean workstations, and physically separate your pre- and post-amplification. B. Double-check your primers are specific to your target sequence and are free from self-annealing, which could lead to non-specific amplification. C. Ensure your magnesium sulfate concentration is within the ideal range for your reaction. D. Try repeating the gel electrophoresis with a known positive sample. Run the gel at a slower voltage to ensure the DNA is not running off the end. Also, check your gel and buffer preparations.

Regards,

Malcolm Nobre

Thank you very much Malcolm Nobre. I'll follow the advice you gave me. I can tell you that I took the oligos from previous publications and tested them by PCR. I do have my negative control, which shows no color change and does include DNA and oligos. We've already standardized the sulfate concentration. I just need to make corrections to the electrophoresis.