Going to the limits of multiplex qPCR

Abstract
Disease with the same symptoms can be caused by up to two dozens different pathogens. Successive single pathogen testing is too slow; parallel testing costs too many wells and is too expen- sive. Multiplex PCR testing increases the throughput and reduces the costs per assay and target while providing at least a semiquan- titative result, helping to rate the significance of the results and to identify putative contaminations.
Some pathogens can cause different diseases. We combine single target assay modules in one multiplex reaction allowing customized combinations for multiplex testing using Roche 480 instruments.
Multiplex realtime qPCR is limited by the number of instru- ment dye channels but also by molecular interferences. Highly multiplexed PCR assays are therefore limited in their sensitivity, in particular if run as 1-step RT PCR on RNA. Stategies to weaken primer binding during the reverse transcription step increases the PCR perfomance.
For testing on bacterial resitance we combine routinely 10 tar- gets in one well, reaching a sensitivity of significant less than 10 copies per reaction. In the Respiratory Viral Panel we combine rou- tinely 11 targets in one well, achieving for the most targets still 10 copies per reaction.
http://dx.doi.org/10.1016/j.bdq.2017.02.049