Dear Oniha

I am attaching a link below which will helpful for you and if there are remaining any problem then you can contact me at any time without any hesitation.

 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564153/pdf/ppj-31-278.pdf

Margaret

There are to many primers already available. I think you should try first with them. To take a decision you should know:  Do you know the the genera or family of fungi involved? What region (s) you you want to amplify? In your DNA sample there is a mixture of plant-fungus DNA? In your DNA sample there is a mixture of several fungi?

Thanks for your answers and the link sent to assist with the identification. But I am still very much interested in the designing of my mold fungi for specific characterization of the isolated pathogens.

The most frequently used primers for filamentous fungi are ITS4 and ITS5. I would first use them. You should find a good database to compare your results against in GeneBank. Using frequently-used primers can be advantageous due to the size of the databank already built by other researchers. Good luck!

If you want to design your own species-specific primers first you need the dna sequences, either generated by your self or downloaded from genbank. Then you have to align them and use any of the available programs to produce the primers. I use Geneiuos it has a very easy to use primer design module.

Once you have a series of potential primers you have to test them agains genbank to see if they only amplify the target species

If you are interested in only ITS sequences, then you really dont need to design primers. You can try using ITS1 and ITS4 or ITS1F and ITS4 for higher fungi (ascomycetes and basidiomycetes). These should work for most fungi. If you decide to amplify other gene regions, more primer design tools for species-specific fungi include https://lifescience.roche.com/webapp/wcs/stores/servlet/CategoryDisplay?tab=Assay+Design+Center&identifier=Universal+Probe+Library&langId=-1 and http://bioinfo.ut.ee/primer3/