I am working with two types of viruses from the same family. I was provided with specifically designed primers to test if either of the viruses are present in the material. However, I ran a PCR and it shows all my samples contain both viruses according to the primers I used. All those samples must have only one virus! The fragment size is exactly the same as it needed to be if there would be another virus.
There are two possibilities: either my starting material was infected with both types of the viruses (or I contaminated them during the process) OR the primer is not specific for only one type of the virus since both of them are quite similar (the same family).
I did PRIMER-BLAST: no, all my primers 100% fits only my desired type of virus and nothing else.
I did BLAST-TWO-SEQUENCES - Highly similar sequences (mega-blast) - comparing both of the types of viruses: yes, it finds 3 places where it is similar but it looks like none of the places fits my primer sequence.
How can I analyse those results? Maybe there is some better way to check my primer?
How does it work?
I have forward and reverse primer, they have a specific sequence for exact regions of some organism. Can they attach to somewhat similar sequence and do their job? If yes then what are the minimum requirements for the attachment?
Primers only need to find complementarity at the 3' end to start PCR. As small as 4-5 bases will be enough binding. As long as you have the 3' end binding in both primers, PCR will proceed. Do you think this is the reason for your results?
Thank you! I will check now only the 3' ends to see if it was the problem. Meanwhile, I decided to run another PCR using different primers. If the result will be positive again I doubt the problem is in similar DNA... Probably my starting material is just infected with both viruses and I might need to look for another source.
Simple suggestion
put ur sequence of both viruses in APE plasmid editor software and use searche tool puting ur primer sequence and validate whats gone wrong
May be use a different primer set for both viruses to reconfirm their presence. I hope that will help
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