5 Prospects

The implementation of eDNA methodologies for amphibian surveillance encompasses a sequence of five primary stages: sample acquisition, DNA extraction, PCR amplification, sequencing, and data analysis (Figure 1). It is evident from this review that the heterogeneous array of research objectives often engenders considerable divergence within the procedural framework. Noteworthy variables encompass the volumetric scale of aquatic samples, ranging from 15 mL to 10 L, the selection of filter pore sizes spanning the interval from 0.22 μm to 1 μm, and the deliberate designation of genetic loci such as 12S, 16S, and Cytochrome b, each of which shows pronounced heterogeneity. This intricate landscape precludes the facile derivation of a universally ’omnipotent’ experimental protocol. It should be noted that attempts have been made to propagate standardized methodologies for eDNA techniques (Bruce et al., 2021; De Brauwer et al., 2022; Minamoto et al., 2021), however these so far have not encountered widespread acceptance or practical implementation (Takahashi et al., 2023).
The multifaceted paradigm underlying eDNA application, as discussed in section 2 of this review, illustrates the causes for the limited applicability of ’standardization guidelines’ across diverse contexts. The unique physicochemical characteristics of different environments make it necessary to carry out iterative adjustments to the experimental approach blueprint and the methodology, as suitable to varied research goals (Pawlowski, Apothéloz-Perret-Gentil, et al., 2020; Pawlowski, Apothéloz‐Perret‐Gentil, et al., 2020; Taberlet et al., 2018; Takahashi et al., 2023).
Because spatial and temporal differences have a large and unavoidable effect on eDNA capture, it may be possible to improve the accuracy of eDNA methods for investigating species diversity or richness of plants and animals by combining them with distribution or occupancy models.
A high-quality and accurate reference database is required for eDNA metabarcoding, and the information base must be sufficient to cover all species in that experimental region. Errors or gaps in the database will lead to a decrease in the accuracy of the findings (Abad et al., 2016; Šigut et al., 2017; Yang et al., 2017). Continued enrichment and improvement of DNA barcode databases in the future are therefore desirable to improve their accuracy and credibility.
Despite the convenience, accuracy and low cost of the eDNA method, it has many disadvantages compared to traditional surveys, such as the inability to directly observe the life stages and disease conditions of surveyed organisms or to measure organism indicators. eDNA metabarcoding cannot completely replace traditional survey methods, and choice of survey methodology should be weighed against the advantages and limitations of both types of approach, as well as specific research objectives. Complementary use is most likely to achieve the most desirable research results.