In the realm of conservation genetics, numerous DNA collection and genetic analysis methodologies have emerged to support wildlife preservation endeavors. The term environmental DNA (eDNA) denotes the genetic material sourced from inorganic settings, such as ponds, rivers, and oceans, rather than from the organism itself (Minamoto et al. 2012). eDNA analysis provides invaluable insights into conservation genetics, particularly for aquatic organisms (Minamoto 2022). Effective wildlife conservation requires precise information regarding population presence, absence, and density. For aquatic organisms in particular, where individual observation and capture pose challenges, eDNA analysis emerges as a valuable assessment tool. Conventional eDNA analysis typically entails collecting water samples in the field or employing filter paper to capture DNA during filtration, followed by transportation to the laboratory for DNA purification. The purified DNA template is then used for positive/negative determinations through PCR or real-time PCR (Q-PCR), or for metabarcoding analysis using next-generation sequencers (Uchida et al. 2020; Inamoto et al. 2024).
Recent years have seen an escalating demand within eDNA research for on-site genetic testing methodologies that yield results immediately. Positive outcomes from the eDNA analysis of a specific species can prompt an immediate decision to conduct a habitat survey, whereas negative outcomes may allow for swift adjustments to the survey site. Portable Q-PCR instruments that can also be used for eDNA analysis in the field are available (Doi et al. 2021). Nevertheless, this approach encounters challenges, such as high costs of equipment and reagents, along with limitations in the simultaneous processing of multiple samples.
Loop-mediated isothermal amplification (LAMP) is a gene amplification technique that is distinct from PCR-based methods and operates on a unique principle (Mori et al. 2013). LAMP employs four to six primers for DNA amplification, with reactions conducted at an isothermal temperature of approximately 60°C. Magnesium pyrophosphate, a byproduct of the LAMP reaction, can be detected and quantified in real time instead of the DNA product. This turbidity LAMP method exhibits sensitivity and specificity equivalent to those of Q-PCR, coupled with the advantages of cost-effectiveness and a reaction time of less than 60 min (Notomi et al. 2015). In addition, the visual LAMP method provides visual confirmation of positive and negative results by the addition of a visible fluorescent reagent (Fischbach et al. 2015). The LAMP method has been used for genetic testing in diverse studies (Mori et al. 2013; Notomi et al. 2015). However, its application in eDNA research remains limited. In Japan, the giant water bug Kirkaldyia deyrolli inhabits freshwater ecosystems, such as ponds, streams, rice paddies, and rivers (Ohba 2019). Recently, these habitats have disappeared or become fragmented, resulting in metapopulations. Consequently, K. deyrolli is protected under the Act on the Conservation of Endangered Species of Wild Fauna and Flora. However, the status of scattered habitats remains poorly understood because of the rapid population decline of giant water bugs. Employing DNA-based genetic testing methods is deemed more efficient than direct observation to investigate the presence or absence of fragmented aquatic habitats, such as those of giant water bugs (Ogata et al. 2023; Suzuki et al. 2023). In particular, the low-cost LAMP method, which can process multiple samples within 60 minutes, can provide a solution to this challenge. For example, if positive or negative tests for K. deyrolli from eDNA can be conducted rapidly in the field, they can facilitate prompt decision-making, such as adjusting the survey site.
Here, we have developed an eDNA analysis method using a visual LAMP technique in the field, which does not require expensive equipment or facilities and can be executed using easily sourced, commercially available electrical appliances and research reagents.