*2.4.1 The challenge being addressed*

Environmental DNA (eDNA) is defined as the genetic material obtained directly from an environmental sample such as water, soil and sediment. The use of eDNA assessment is well established for detecting the presence or absence of rare or invasive species from the organic material they leave behind [40]. An organism provides a rich source of eDNA through the cells and waste they shed and excrete including faeces, mucus, gametes, hair and skin [41]. eDNA has been used to monitor a wide variety of species in a diverse range of environments.

There are two main approaches to eDNA monitoring: targeting specific species or detection of multiple species from a single sample, termed eDNA metabarcoding [42]. Monitoring of specific species, particularly endangered or invasive species, is vital for conservation purposes. The sensitivity, and importantly specificity, of molecular techniques has enabled the non-invasive monitoring of individual species from a variety of environmental samples [43]. Due to the low concentration of eDNA, these techniques require specific amplification of target DNA to a detectable limit [43]. For this reason, quantitative PCR (qPCR) is most commonly used and through robust primer design, has been shown to enable monitoring of several target species [44–47]. Moreover, qPCR has the potential to be adapted for any target of interest. Although the gold standard, other nucleic acid amplification techniques also offer potential for use in single species detection [48, 49]. For example, isothermal amplification techniques

**Figure 5.**

*Scheme showing the methods used to collect and analyse eDNA.*

### *Water Quality Monitoring Using Innovative Technologies DOI: http://dx.doi.org/10.5772/intechopen.105162*

such as Recombinase Polymerase Amplification (RPA) and Loop-Mediated Isothermal Amplification (LAMP) have been used for diagnostics in the medical field [50] highlighting their specificity and sensitivity. These methods rely on enzymatic activity to maintain DNA amplification efficiency without the need for thermal cycling. On the other hand, eDNA metabarcoding allows identification of multiple species across all taxa from microbes to higher vertebrates as illustrated in **Figure 5**.

## *2.4.2 The sensing approach*

The aim of this work is the development of a biosensor device for single species monitoring that can enable eDNA technology to be taken out of the laboratory and into the field. Conventional methods for eDNA detection pose a logistical challenge for on-site monitoring and adaptation to biosensor devices, due to the need for high temperatures and thermal cycling. Isothermal amplification methods have evolved as an alternative to PCR, enabling nucleic acid amplification at a constant temperature [51]. These methods can be coupled to fluorescence-based detection to enable a highly sensitive and specific analysis of target sequences [52, 53]. An alternative molecular eDNA approach, coupling isothermal RPA to a CRISPR-Cas12a detection system, has been developed.

In this work, three salmonid species were targeted, S. salar, S. trutta and S. alpinus, due to their ecological, economic and cultural importance in Ireland. The developed assays enabled detection of these species at a single temperature of 37°C. This switch from thermal cycling to isothermal detection enhances the potential for adaptation to a biosensor device. This approach has been a transformation in terms of how eDNA sensing in the field can be delivered. The CRISPR-Cas system, more commonly known for its role in genome editing, enables highly specific sequence recognition [53], which can be adapted to detect any species from eDNA samples from a variety of sources. This improves the ability to differentiate closely related species and enhances the capabilities of eDNA as a tool for biodiversity monitoring [54]. This work has for the first time demonstrated the detection of Atlantic salmon eDNA in the presence of other potentially competing species. This exciting development using the CRISPR-Cas system has meant that a sensor can be developed for eDNA target species and used directly in the field providing real-time or near real-time occurrence data.
