I am running 2 multiplex reactions of 5 different prey species that are of genus level relatedness. I have developed species specific primers for 4/5 species, but the 5th species primers always amplify the predator DNA at the exact same size of the expected prey product size. I have tried at least 6 different primer sets and every single one creates a product for the predator that is equal to the prey.
There is no way the predators could have eaten any of this prey species and I have extracted the DNA of multiple predators to be sure there wasn't any contamination. I am pretty new at PCR and don't understand how a very unrelated species could be amplified when other extremely closely related prey species don't amplify the predator DNA.
I guess my main question is why the primers would amplify the predator DNA to a product identical to the desired prey species every time.
I have been trying to troubleshoot this for over a month now and have run out of ideas besides sequencing the predator DNA but the situation just confounds me.