We report the first application of CRISPR‐Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows noninvasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR‐based techniques to detect a given species from eDNA samples, posing a logistical challenge for on‐site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR‐Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof‐of‐concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 hr). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity.
<p>The discharge of phosphorus associated with wastewater has decreased significantly in Europe over the past 25 years<sup>1</sup>, however the problem of diffuse pollution persists<sup>2</sup>.&#160; Studies have shown that regulatory monitoring can miss elevated spikes in phosphorus concentrations<sup>3</sup> and high frequency monitoring is required<sup>4</sup>. Such programmes are resource intensive, requiring effective tools which enable appropriate water quality data collection and quality assurance<sup>5</sup>.</p><p>A low cost, portable, and rapid phosphate detection system is needed to enable the quick detection of phosphate in areas affected by high phosphate levels<sup>6</sup>. A new system is being developed by evolving a colorimetric detection system using microfluidic lab-on-a-disc technology which has previously been demonstrated<sup>7</sup>. It utilizes a micro-spectrometer and the molybdenum blue method, and has been built with the intent of requiring limited training.</p><p>The range of the system is 5-400 &#181;g/L, which encompasses the threshold value of 35 &#181;g/L P for Irish rivers and groundwaters<sup>8</sup>. The system is extremely portable due to its compact size and weighing less than 2 kg. With a run time of 15 minutes per ten samples, it enables the in-situ detection of phosphate for rapid on-site monitoring.</p><p>To test the system, rivers in the northwest of Ireland were identified. Three of these rivers have historical orthophosphate readings in the range of 5 - 47 &#181;g/L and two others were reported considerably higher at 84 &#181;g/L.&#160;&#160;</p><p>With this microfluidic phosphate detection system, rapid in-situ detection and reliable, real-time monitoring of phosphorus in freshwater systems can be achieved.&#160;</p><p><strong>References:</strong></p><p>1)<em>European waters -- Assessment of status and pressures 2018 &#8212; European Environment Agency</em>. https://www.eea.europa.eu/publications/state-of-water (accessed 2022-06-13).</p><p>2)Biddulph, M.; Collins, A. l.; Foster, I. d. l.; Holmes, N. The Scale Problem in Tackling Diffuse Water Pollution from Agriculture: Insights from the Avon Demonstration Test Catchment Programme in England. <em>River Research and Applications</em> <strong>2017</strong>, <em>33</em> (10), 1527&#8211;1538. https://doi.org/10.1002/rra.3222.</p><p>3)Fones, G. R.; Bakir, A.; Gray, J.; Mattingley, L.; Measham, N.; Knight, P.; Bowes, M. J.; Greenwood, R.; Mills, G. A. Using High-Frequency Phosphorus Monitoring for Water Quality Management: A Case Study of the Upper River Itchen, UK. <em>Environ Monit Assess</em> <strong>2020</strong>, <em>192</em> (3), 184. https://doi.org/10.1007/s10661-020-8138-0.</p><p>4)Bowes, M. J.; Palmer-Felgate, E. J.; Jarvie, H. P.; Loewenthal, M.; Wickham, H. D.; Harman, S. A.; Carr, E. High-Frequency Phosphorus Monitoring of the River Kennet, UK: Are Ecological Problems Due to Intermittent Sewage Treatment Works Failures? <em> Environ. Monit.</em> <strong>2012</strong>, <em>14</em> (12), 3137&#8211;3145. https://doi.org/10.1039/C2EM30705G.</p><p>5)Quinn, N. W. T.; Dinar, A.; Sridharan, V. Decision Support Tools for Water Quality Management. <em>Water</em> <strong>2022</strong>, <em>14</em> (22), 3644. https://doi.org/10.3390/w14223644.</p><p>6)Park J.; Kim, K. T.; Lee; W. H. Recent advances in information and communications technology (ICT) and sensor technology for monitoring water quality. <strong>2020</strong>, <em>Water, 12</em> (2)</p><p>7)O&#8217;Grady, J., Kent N., Regan, F. (2021). Design, build and demonstration of a fast, reliable&#160; portable phosphate field analyser. <em>Case Stud. Chem. Environ. Eng.</em>, <strong>2020</strong>, <em>4</em>, 100168</p><p>8)Tierney, D.; O&#8217;Boyle, S. <em>Water Quality in 2016: An Indicators Report</em>; Environmental Protection Agency, Ireland: Wexford, 2018; p 48.</p>
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