Hi, I have a collection on isolates we are going to inoculate in plants. To quantify them in planta, we want to design strain-specific primers for RT-qPCR.
What I did till now:
Since I have all the genomes from my isolates, based on pan-genomic analysis I extracted a core gene (found in all isolates, coding for MACP) with low level of homogeneity. I have all those MACP sequences from my isolates in a multifasta, but I could not find the right tool to design a primer from them.
One way would be to input them 1 at the time in BLAST-primer, but there are no promises of course regarding the strain-specificity of the primers?
Otherwise I could align them and look for flanking conserved regions to design the primers on. But that would require sequencing afterwards to determine which strain they belong to, right?
Do you have any suggestions how to approach the problem?
If you have any other idea regarding strain-specific primer design or know some good tool, I would be happy to hear them. Until now SpeciesPrimer gave me only 2-3 ssPrimers from my 88 isolates and I could not manage to get find_differential_primers running yet.