Design of FRET-TaqMan probes for multiplex real-time PCR using an internal positive control.

The multiplexing capabilities with different fluorescent dyes are limited in real-time PCR instruments equipped with one excitation source. Considering this limitation, a design was developed to create a triple-labeled probe as an internal positive control (IPC) that utilizes a combination of the fluorescence resonance energy transfer (FRET) and TaqMan techniques. The IPC probe, labeled with FAM and Cy5.5 fluorophores at the 5' end and Black Hole Quencher (BHQ) at the 3' end, enabled Cy5.5 emission through energy transfer from the FAM fluorophore. The second, target-specific TaqMan assay in the multiplex used a FAM- and BHQ1-labeled probe at the 5' and 3' ends, respectively. Thus, one excitation source was used to generate two different fluorescence emissions (FAM and Cy5.5) that were measured in two separate channels by the real-time PCR instrument. This method can facilitate the development of a low-cost portable handheld real-time PCR instrument capable of multiplex real-time PCR assays using a single excitation source.