Multiplex Real-time PCR

Multiplex PCR refers to the use of more than one primer pair in one reaction tube in order to amplify more than one target sequence or to more than one primer/probe set in the case of real-time PCR. The internal control assay is one example of multiplex PCR. In microbiological diagnostics, the general aim of multiplex PCR is to test simultaneously for different agents in the same reaction. This requires the use of fluorophores and equipment with the ability to distinguish between the different assays. Non-specific amplification products seem to be the most limiting factor of multiplex PCR as the number of oligonucleotide primers and probes increases. Several multiplex assays for the simultaneous detection of two or more different agents are, however, well established. Examples include multiplex PCR assays for herpes simplex virus 1 and 2 (HSV-1 and HSV-2) and assays for influenza type A and B viruses.