Identifying Snow Leopards, Tigers and other Endangered Species by developing a low-cost DNA field test kit - Part 2

Methods

Summary

The method will adapt a paper-based biosensor technique developed by a biomedical lab at McMaster University for the early detection of disease and E.coli bacteria in food and water using rolling circle DNA amplification coupled with a colorimetric response. Rolling circle amplification (RCA) is an isothermal enzymatic process where a short DNA primer is amplified to form long single-stranded DNA using a circular DNA template and a special DNA polymerase (Ali et al. 2014). The power, high specificity, simplicity and versatility of the technique have made it an attractive tool for the detection of DNA, particularly DNA of low quality and concentration. Alternative isothermal DNA amplification techniques such as LAMP require complicated designs of multiple primers producing even more complicated DNA structures of various sizes which make them sensitive to error (Karami et al. 2011). Furthermore, RCA was found to be more proficient on paper than in solution compared to other single temperature amplification techniques (Liu et al. 2016). The same paper can also be used for DNA extraction and denaturation, reducing the total number of steps and handling from animal remains to colorimetric signal.

The method will be tested in the field at various snow leopard hotspots around Mustang in Northern Nepal and later verified using traditional DNA profiling methods at the Centre for Molecular Dynamics. In parallel to the method verification, we will strategically interview local people from each community with the goal of developing initiatives to financially empower them and change their attitudes towards snow leopards in the hope that they can in turn support monitoring programs in the future.