If you’re testing whether a single species is present or not, what is the advantage of using qPCR over PCR? And qPCR versus Metabarcoding?
qPCR (probe-based) assay is often more reliable than PCR (end-point PCR visualised on a gel) because the binding of the probe represents a third point (in addition to the two primer sequences) at which the target sequence must be matched, thereby reducing the risk of non-target amplification causing false positive results . qPCR is much faster to carry out in the lab than metabarcoding, which requires a substantial amount more lab work and computational processing. However, because qPCR and other types of single-species screening assay are ‘blind’ tests which give a positive or negative result without providing a sequence to confirm species identity, the assays need to be extremely rigorously validated before you can rely heavily on the results for decision-making in management contexts (Thalinger et al., 2020 gives a comprehensive overview of this). This can take a long time and is an expensive process. Metabarcoding can provide data on many more species than qPCR, and because it generates the DNA sequences that are used for determining species identity, high confidence can be ascribed to detections even early on in the assay validation process.
