I am currently using a conventional PCR with a probe based assay (reverse line blot) to identify species of nematodes. I am using universal primers and I have noticed that some species template DNA is favored over another species in a mixed sample during the PCR even when it is present in far lower concentrations than the non amplified species. I know that one species is being favoured because when I amplify either template on there own I get a positive result on my reverse line blot, but not when they are mixed. The universal primers are an exact match for both species and the amplicon is roughly the same size, at most a 20 bo difference also the sample qualities are very similar in regards the 260/280 ratio.
I have sequenced the different species myself and I am sure that the primers are an exact match for all species
Can anyone help explain why one species template is favored over another when using universal primers in a mixed sample.