Significant research e↵orts are continually being directed towards the development of sensitive and accurate surface plasmon resonance biosensors for sequence specific DNA detection. These sensors hold great potential for applications in healthcare and diagnostics. However, performance of these sensors in practical usage scenarios is often limited due to interference from the sample matrix. This work shows how the co-immobilization of glycol (PEG) diluents or “back filling” of the DNA sensing layer can successfully address these problems. A novel SPR based melting assay is used for the analysis of a synthetic oligomer target as well as PCR amplified genomic DNA extracted from Legionella Pneumophila. The benefits of sensing layer back filling on the assay performance are first demonstrated through melting analysis of the oligmomer target and it is shown how back filling enables accurate discrimination of Legionella Pneumophila serogroups directly from the PCR reaction product with complete suppression of sensor fouling.