Guidlines for species specific probe design

[brendanconneely]

Hello,
Im trying to design a panel of species specific probes for the identification of 13 species. The probes will be employed on a reverse line blot (which is very similar to a micro array only larger and cheaper). I am using the internal transcribed spacer 2 (ITS2) region of the rDNA array as a diagnostic target. My problem is I dont have any guidelines on the probe design only that they should be between 18-30 bases in length. My main question is how many bases in my probe sequence need to be need to be different from other species ITS2 sequence to make that probe species specific, and also where should these differences occurr in my probe sequence, the outside of the sequence or in the middle ?
Any help would be greatly appreciated