Animal species detection by polymerase chain reaction (PCR) is based on the amplification of a specific region of the relevant species genome. In real-time PCR, the amplified product is detected via target-specific fluorescent probes that bind to the amplified product. Accumulation of PCR product results in increased fluorescent signal from the bound probes. Monitoring the fluorescence intensities during the PCR run (i.e., in real time) allows the detection of the accumulating PCR product without having to re-open the reaction tubes after the PCR run.
The probes of mericon PCR Assays are sequence-specific oligonucleotides with a fluorophore and a quencher moiety attached. The fluorophore is at the 5' end of the probe, and the quencher moiety is located at the 3' end. If the target DNA sequence is present, the probe is cleaved by the 5'-->3' exonuclease activity of HotStarTaq Plus DNA Polymerase during the extension phase of PCR. This separates the fluorophore and the quencher moiety, resulting in a detectable fluorescence that is proportional to the amount of accumulated PCR product.
The PCR primer set for each assay is highly specific and targets a unique and conserved DNA region of the tested species genome that is verified bioinformatically and experimentally. Cross-reactivity has been bioinformatically investigated and thoroughly tested with a panel of selected targets for each mericon PCR Assay. Each assay can detect as few as 10 target copies of each target in a reaction.