Hello everyone.

We are trying to design a multiplex qPCR to detect three different viral sequences, by using three pairs of primers and three different fluorescent quenched probes:

1) HEX - BHQ-1

2) Texas Red - BHQ-2

3) Cy5 - BHQ-2

Before joining all three in a multiplex PCR, we tested the performance of each probe separately. The problem is only the first sequence (probed with HEX - BHQ-1) is being detected.

The other two probes seem to have no fluorescence at all, as the positive controls for those sequences behave just like the negative control (zero signal). In fact, our LightCyler 2.0 (Roche) cannot even detect the reaction capillaries (which happens when there is too little fluorescence).

We do know that amplification takes place. We can see a band of the right size after running the product in an agarose gel.

In the past, we have successfully designed qPCR probes for the simultaneous detection of different pairs of sequences, but we have always used BHQ-1 as a quencher. What do you think the problem could be? Is it the BHQ-2 quencher? Are Texas Red and Cy5 fluorophores not suitable for our instrument? Thanks for your answers.

Carlos.