Traditional PCR and assays’ multiplexing
The possibility to visualize the assays’ outcomes using the traditional
PCR, rather than qPCR, is a desirable feature, as it allows the test to
be carried out in any structure equipped with essential molecular
biology laboratory apparatus, besides being cheaper. Although the
diagnostic sensitivity decreases in the case of traditional PCR, our
study indicates that when the signal is not too weak (see below), even
basic PCR allows the identification of traces of monk seal DNA. The
possibility of using the traditional PCR, in turn, entails further
advantages, such as the possibility of multiplex run, particularly
advantageous when multiple assays are available. This procedure reserves
more plusses. Firstly, it allows saving on the amount of eDNA template
(precious as typically limited) to be used. Another advantage is the
possibility of amplifying the template molecule with different probes at
once, a strategy that may reveal useful when dealing with highly-diluted
eDNA of the desirable target species and especially for large body size
species which are likely to shed “clustered” eDNA (e.g. only one of
the three replica-filters, may contain the target species DNA, although
they all come from the same water sample). In these instances,
multiplexing could minimize the possibility of false negatives while
possibly gathering clues about the signal age (see above), although the
latter aspect needs further deepening. A preliminary multiplex test
indicates that all combinations of the three proposed assays are
compatible for multiplexing and that the approach is efficient also with
eDNA samples, providing a relatively high (say >10
log2DNA copies/liter) monk seal DNA content such as in
Madeira control sample MmoM+01 (Figure S5).