I'm working on CCCVd molecular detection work.
Expected RNA present very low concentration in samples ( ≤ 1 μL per sample). When following nested PCR, at first PCR unable to get the expected size band and at the second PCR band appears (image attached).
What will be the routine diagnosis full length sequence of this viroid?
You could try some cheap options such as
1. Increase the number of cycles in first cycle of nested PCR, if not, try second option
2. You could try with double stand RNA extraction since double standard are highly stable than single stranded, and quite often works for some RNA viruses. It works for both single stranded RNA and ds RNA.
Check the paper
http://www.apsnet.org/publications/phytopathology/backissues/Documents/1979Articles/Phyto69n08_854.pdf
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