I'm designing a diagnostic qPCR triplex assay for three parasitic species and have this weird weird thing that keeps happening where I test my assays in singleplex and they're specific but then in triplex one of them isn't. I have done every possible BLAST and bioinformatic analysis to verify that the assay isn't cross amplifying, so I'm certain it's not a specificity issue. There's no genomic explanation and I've also contacted both ThermoFisher (the maker of the machine) and IDT (where we get our primers) and they said there was nothing that should be causing this problem.

The three probes I'm using are FAM, VIC, and TAMRA - recommended by a ThermoFisher rep and our machines are calibrated for all of these (I've seen this weird thing happen on multiple machines). The VIC assay is picking up the FAM target at almost the exact same Cq values as the FAM assay is picking up the FAM target. To me, that means the two species would have to be present in equal parts in the sample and it's not just a contamination problem since I'm putting in just one species' DNA in each well. The VIC assay still picks up the VIC target, but LATER than it picks up the FAM target. So apparently the VIC assay is picking up a completely unrelated species far better than it's own target species?? I think not.

I've done so much to verify that it's not a specificity issue, including running the VIC assay in singleplex with the FAM target DNA and only two wells of a positive control to make sure the qPCR worked - it showed absolutely no amplification in any of the FAM target wells. My NTC wells are always negative and my positive controls always work. I've reached out to anyone I can think of for help because I don't know what on earth is going on and no one has been able to give me even any possibilities. I have this weird feeling it's something with the settings on the StepOne Plus, but I'm not sure what.

Has anyone else had anything like this happen? Or does anyone have suggestions of other things to try?