Structures of the immuno-polymerase chain reaction (PCR) Multiple Simultaneous Tag (MUSTag) components.

A. The MUSTag assay consists of 3 parts: a capture antibody, a recombinant protein G-avidin fusion protein, and a synthesized biotin-conjugated oligonucleotide (A). B. The MUSTag assay is performed similar to the sandwich enzyme-linked immunosorbent assay (ELISA) but by using a biotin-labeled detection antibody instead of an enzyme-labeled antibody. C. MUSTag oligo-DNAs were designed to incorporate 5 defined sequence regions: the 5′ primer for template amplification with biotin labels on sequence (F1 primer) and the complement of the 3′ primer (R primer), the 5′ primer for qRT-PCR detection (F2 primer) with R primer, an EcoRI restriction site, and, for hybridization with fluorogenic TaqMan, a double-labeled hybridization probe that annealed between the F2 primer and the R primer. D. The complement sequences of the F2 and R primers and TaqMan Probe were used for qRT-PCR measurement of the original concentration of GLA.