Multiplex Molecular Reactions: Design and Troubleshooting

Multiplex molecular assays are powerful tools that can provide rapid solutions to challenging diagnostic questions. A number of different technologies have been successfully used for multiplex molecular detection. Each method has its own set of unique advantages and drawbacks, all with an inherent challenge to maintain assay sensitivity in the presence of potentially cross-reacting and competing amplification or target detection. A central feature of virtually all multiplex molecular methods is the polymerase chain reaction (PCR). Knowledge of the requisite steps for effective development and optimization of PCR is needed to overcome multiplexing challenges and to successfully create and troubleshoot multiplex assays. This review will discuss key points of assay design and optimization, with an emphasis on how this knowledge can aid in the implementation and ongoing troubleshooting of multiplex molecular assays.