I'm on a team designing a multiplex PCR assay to use to identify bacterial pathogens. We have a set of 6 primers and probes that we can use to identify 9 different species of this particular bacteria. However, we're running into an issue where in our second Multiplex, the fluorescence from the FAM channel is 'leaking' into our Hex channel and giving us false positives.

So far we've been running a third control of JUST the FAM primer/probe in order to establish a proper baseline but this won't be feasible if we present our multiplex as a diagnostic tool for other laboratories (i.e. needlessly complex is unwanted).

What might be causing that leakage? Would choosing different probes solve the problem? (Hex and FAM are near one another in wavelength emitted) Do the primers themselves need to be redesigned?

I appreciate any and all help!