This would presume equal efficacy for all reactions, but could try to limit dNTP and/or primer concentrations. Once those plateau, the reaction will not commence.

As it is multiplex, limiting the primer concentration seem a good strategy as Péter Gyarmati. Specially if you do a previous "titration" of the optimal primer concentration to know in which CT/Threeshold you reach that "Plateu" and stop the reaction for the desired genes.

I hope that helps!

Best regards,

Francesc Estrany

Péter Gyarmati

I appreciate your response. The difficulty lies in the lack of information regarding template concentrations and the uncertainty surrounding the appropriate levels for primers and dNTPs. To address this, I am considering designing a customized primer and amplicon structure. Additionally, I am investigating different physical or chemical methods to achieve this goal. Once the amplicon reaches a specific threshold, such as 1000 copies, the structure or a specific chemical feature should be implemented to halt the PCR reaction.

Francesc Estrany

The difficulty lies in the lack of information regarding template concentrations and the uncertainty surrounding the appropriate levels for primers and dNTPs. The template would be a clinical specimen and target amplicon would be pathegon microoragnisms, such as bacteria, virus or fungi. Determining the precise levels of each microorganism within the specimens remains uncertain. Balance the PCR efficiency of each primer in multiplex PCR system is challenging, Therefore, I have attempted to implement a strategy where I halt the PCR reaction for each primer individually upon reaching a predetermined threshold.