9.3.7. Multiplex RT-(q)PCR
Multiplex RT-PCR refers to the simultaneous amplification of several targets in the same reaction. The different end-products are usually identified by size, through (gel) electrophoresis. Several such qualitative multiplex protocols have been designed for honey bee viruses (Chen et al., 2004b; Topley et al., 2005; Grabensteiner et al., 2007; Weinstein-Texiera et al., 2008; Meeus et al., 2010). Real-time qPCR can also be multiplexed, usually for the simultaneous amplification of a target and internal reference standards, by using TaqMan™ probes with different fluorophores.
The main reason for multiplexing is to save cost and time. However, multiplex PCR is less sensitive than uniplex PCR, more complex to optimize, more prone to artefacts and requires post-PCR fragment analysis, nullifying any gains in time and cost. Most importantly, the late amplification of low-abundance targets is strongly affected by the prior amplification of high-abundance targets, through the auto-inhibition of the PCR by the DNA it produces (SantaLucia, 2007). For these reasons, it is often more effective to use uniplex RT-PCR, even for large projects.