Because the Bst 3.0 has the function of RTase，so I want to construct the RT-LAMP with RNA template in one step.
1. I confirmed the RNA sample contains the target virus with RT-PCR;
2. I conducted LAMP using the Bst 3.0 with the cDNA，and the result was positive;
3. but when I changed the template from cDNA to the original RNA, and conducted the RT-LAMP using Bst 3.0, the result was negtive.
The difference between “2” and "3" was the template only, but the results were inconsistent. what's the reson and how can I solve it?
My guess would be that your RNA sample is degraded, are you using extracted RNA or synthetic samples? RNA should be stored at -80 degrees for long-term storage and should not undergo more than 3 freeze-thaw cycles. Have you compared freshly extracted RNA samples with the ones you are using right now?
Diogo Figueiredo the RNA was extracted RNA stored at -20℃. Because there is an experience that such RNA can still obtain positive results by RT-PCR after several times of freeze-thaw cycle, so I think its degradation is not so serious, so I did not compare freshly extracted RNA samples with the ones using right now.
Thanks for your reminding, I will perform RT-LAMP amplification again using the RNA stored at -80℃.
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