Here, I want to ask about my issue with the SARS-CoV-2 RPA amplification False-Positive results. I tried two different sets of primers. The first sets of primers are 29 nt producing 200 bp amplicons. The second sets are 33 nt producing 121 bp. Also, I used both liquid and basic form of twist RPA kit. However, I always got False-Positive results in all of my amplifications (Attached figures). I would appreciate it if you have any experience or suggestions to solve this issue?
Hello Reza Nouri ,
According to my experience with the RPA amplification, If you have any doubt about the specificity of the primers, I recommend you test your RPA primers in conventional PCR conditions (using a DNA polymerase). This way, you can sequence your target region and confirm that you are amplifying the viral target region. Despite the big size of the RPA primers, these can be used in conventional PCR condition without any problem. The Sequencing the target region of your primers (29 and/or 33 bp) you can know if you are amplifying the virus or not.
I have see your gels and is possible that your RPA reaction works well. (Some time a clear band is difficult to obtain using RPA amplification). In my opinion, you have to sequence your amplification. This maybe clarifies your "problem".
In addition, if you are developing a new method for viral detection, I suggest you make differents specificity assay, including differents samples with another potential virus that you can find in the host.
I suggest you check this paper... maybe will be useful for you.
If my answer has been useful, please recommend it.
:) :)
Good luck!
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