Cross-species Specific Primer Design with MP-Ref

July 21, 2025

Designing primers with excellent cross-species specificity using MP-Ref (demo link).

1. Select the target database;
2. Enter the targets in BED format;
3. Also select “dog” and “E. coli” as backgrounds databases;
4. And set “Min=0 Max=0” both for “Number of Off-targets” and “Amplicon Count on Background Genomes”;
5. Download the primers and open it in Excel;
6. Copy the primers and paste to MFEprimer for validation: https://m4.igenetech.com/spec/. Select the three databases which we used for designed primers.
7. MFEprimer shows only the three target amplicons as we expected (demo link).