When Blasting primers via the NCBI web site it automatically detects that the query is a short sequence and automatically adjusts the search parameters. Geneious does not do this. However, you can change the default Blast settings in Geneious to improve Blast with primer sequences.
The screenshot below shows the Blast settings you should use. These settings match those used by the NCBI Blast web site when Blasting short sequences.
Once you have changed the default settings, click on the cog in the bottom left of the Blast set up window and choose "Save current settings" to create a Profile that can be used with other primer sequences.
Users should be aware that there are issues with BLAST when searching for short sequences. Blast is not guaranteed to find all occurrences of a short sequence in a database. Statistically, even with the word size set to 7 (the minimum for DNA searches) BLAST will typically miss 40% of possible hits when dealing with sequences of 20bp. In addition to potentially missing a large percentage of possible matches, any matches Blast does produce will be local alignments rather than full length matches.
If you want an alternative to Test with Saved Primers because ‘Test with Saved Primers’ is computationally intense, we recommend using the Map to Reference function rather than Blast. The Map to Reference function is optimised for short sequence matching.
Note that if the primer has a 5’ extension then this should be annotated onto the sequence correctly. Geneious will ignore the extension region when primer testing. If this isn’t done, the primer will not match.