The purpose of this study was to apply an intentional mismatch design technique for primer-based detection of gene targets. In this study, wild-type versus mutant cystic fibrosis transmembrane conductance regulator alleles as well as two related adenovirus B7 and E4 genomes were used to test discrimination of primers. Lambda viral DNA genome template as well as published primers versus purified human genome template was used as controls. The presence of the ΔF508 mutation and adenovirus B7 could be accurately detected using allele-specific primers, with the reverse ΔF508 primers containing an intentional mismatch, to increase the steric hindrance, therefore both diagnosing CFTR and associated viruses.