Fig. 6

Outline of the E-RT-MCDA assay. There are seven steps in this schematic: Step 1, at a constant temperature, the double-stranded DNA becomes scattered single strands, and the different primer binding sites are highlighted. Steps 2 and 5, the ssDNA strands (including sense strands and antisense strands) as templates are amplified in the MCDA reaction system. Steps 3 and 6, at the start of amplification, the short sequence (5'-TGCAATGNN-3') is recognized and cleaved by the Nb.BsrDI enzyme. Steps 4 and 7, the cleavage causes the fluorescein groups (FAM) and the dark quencher (BHQ1) to separate from the two sides of the short sequences, and the fluorescent signals are collected by a real-time fluorescence detector