My project is about detection of species DNA in feed sample. I get a strange melt curve result from a developed species detection assay. The assay was optimized with genomic DNA. Peak at Tm=74 is the target species, peak at Tm=80.5 is the internal control species. Attached is the melt curve analysis.
Positive control gave me two significant peaks. Some blind sample showed an expected peak at 80.5 while few of them gave me not only an expected peak at 80.5, but also an addition peak at Tm=77.0. I tested those blind sample again in separated tube (singleplex) and realized the extra peak is actually derived from internal control.
My question is, whether this duplex qpcr result is valid? Is the extra peak matters?
Please share me your thought. Any opinion is welcomed.