In multiplex qPCR, will the fluorescent groups of different probes interfere with each other?

When designing, don't just look at Tm. The Tm of the three primer pairs should be as consistent as possible, with differences controlled within 2°C; probe Tm can be 5-8°C higher than primers, but the three probes should also be close to each other. Amplicons should be as short as possible, with 80-150 bp being suitable. Serum contains many nucleic acid inhibitors, so short amplicons provide more stable efficiency. Also check for primer dimers and 3' end overlap between probes, especially avoid consecutive G at the 5' end of the FAM probe, which may cause quenching. It is recommended to start all primers at 200 nM and probes at 150 nM. If any channel has low sensitivity, increase it individually rather than simultaneously increasing the total amount of all three primer pairs. During synthesis, have the supplier aliquot by different fluorophores and protect from light.

For serum nucleic acid, load 5 μL and if sensitivity drops, first check for inhibitors. You can run a template dilution series to see whether the Ct difference between triplex and singleplex increases with dilution; alternatively, switch to an inhibitor-tolerant mix. Another key point is the ROX channel setting on the CFX96: if your mix contains ROX as a reference dye, do not use ROX as a probe label, otherwise it will conflict with the reference. On this type of instrument, we generally change to FAM/HEX/Cy5 or FAM/VIC/Cy5 to avoid using ROX. If the ROX probe must be retained, confirm that the instrument is set to no ROX reference and that the mix does not contain ROX, otherwise the baseline will be interfered with.

For CFX96 running FAM/HEX/ROX triplex, it is recommended to first perform singleplex validation, then conduct a duplex cross-experiment: remove one probe at a time and check whether the corresponding channel shows signal, to determine crosstalk. In the FAM/HEX/ROX combination, bleed-through is likely between HEX and ROX, especially when endpoint fluorescence readings are high. If crosstalk is found, first adjust channel gain, threshold, or change the fluorescence combination; do not rely solely on software automatic compensation. Use the same batch of standards to compare singleplex and triplex Ct values; a difference within 0.5-1 cycle is acceptable. If the NTC in a certain channel is elevated, prioritize checking probe concentration and primer dimers.