Dear researchers,

I’m working on an Norovirus GI and GII qPCR assay with TaqMan probes. Recently, I experienced an issue with “multiplexing”. Briefly, the aim is to develop an assay for simultaneous detection of two targets (HuNoV GI and GII) using FAM and HEX reporters.

Single PCR, when the probes are used separately, works well. While in multiplex (when four primers, two probes are mixed per reaction), we get a low signal for one of the targets in several independent runs. On the other hand, in electrophoresis the DNA yield seems the same for different combinations. I tried to change probes concentrations and it seems that reducing probe amount for one target increases signal for the other target, but I guess that it may affect my results for competing target. I would appreciate any suggestion to optimize this assay, including good manulal about assay development. What to do in this kind of situation?

Thank you!

Technical details:

Concentrations of primers 400 nM, probes 200 nM, Mg 3 mM.

Cycling conditions: hot start at 95⁰C for 5 minutes, then 45 cycles of denaturation at 95⁰C for 15 seconds, annealing and elongation at 60⁰C for 1 minute.

Instrument: ABI StepOne Plus