Figure 1.
Schematic representation of the Multicolor Combinatorial Probe Coding (MCPC) strategy for multiplex, real-time PCR genotyping using displacing probes.
Four differently colored fluorophores (FAM: green; HEX: orange; ROX: red; and Cy5: blue) and one universal quencher (DABCYL: black) are used to depict the principal of MCPC. A) Illustration of how half the molecules of a third probe in an assay can be labeled in one color and the other half of the molecules of the third probe can be labeled with a second color, producing a single probe that generates a two-color signal when it hybridizes to its target. B) Four probes, each labeled with a different single color. C) Six probes, each labeled with a unique combination of two of the four colors. D) Four probes, each labeled with a unique combination of three of the four colors, and one probe labeled with all four colors. For each fluorophore, a unique four-digit code, MCPC signature, is given, i.e., FAM is 1000, HEX is 0100, ROX is 0010, Cy5 is 0001. For the combined fluorophores, the MCPC signatures are also combined for coding.
Figure 2.
Real-time PCR typing of HPV with MCPC.
One pair of primers and 15 probe sets were mixed in each reaction tube for identification of one of the 15 possible viral types in a real-time PCR setting, as described in the Material and Methods section. Fluorescence signals were recorded in all four channels of the fluorometric thermocycler (FAM: green; HEX: orange; ROX: red; and Cy5: blue). The MCPC signature is given for each type.
Figure 3.
Representative results of real-time PCR quantification with MCPC for S. aureus, V. cholerae, Shigella spp. and S. typhi.
Purified DNA templates were 10-fold serially diluted from 100 ng to 100 fg and water was used as non-template control. The MCPC signature is given for each bacterium type. FAM: green; HEX: orange; ROX: red; and Cy5: blue.
Table 1.
Quantitative detection of 10 bacterial strains by MCPC in a single tube.
Table 2.
Twenty-one possible genotypes and their MCPC signatures.
Figure 4.
Real-time PCR genotyping of 13 different β-globin genotypes in clinical samples with MCPC.
The assigned genotypes are listed with MCPC signatures and genotypes.