Dear all,
I am doing an RT-LAMP technique for RNA Tembusu virus detection. I used pre-adding HNB for the direct detection by the naked eye. In the beginning, the RT-LAMP reactions seem to work well. Then it began to give strong false positives with negative samples and controls. I did not open the tubes after amplification. I checked the contamination by RT-PCR but it's negative. Then I reduced the amplification time to avoid non-specific amplification. However, the results are still not stable. Even, I mixed new reagents for RT-LAMP and I tested with another RNA template, it works well. But, for the RNA Tembusu virus, sometimes negative control is negative and some are positive. I read some papers suggested that using DMSO to reduce the non-specific amplification. But the results were negative (including positive and negative control)...
I don't know what happened. Please help me with any suggestion about that.
Thank you very much.
I have a lot of experience using RT-LAMP for CoV2 testing via saliva as the transport medium. The materials I used (WarmStart by NEB) would drop in pH resulting in the visible change in color as it used phenol red as the indicator. But, a small percentage of the population has saliva with a pH low enough to give false positives anyway. If you are using a visible version LAMP product and you are having these discrepancies I would suggest looking into the pH as the culprit. Hope this helps!
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