Comparison of results between the field-concentrated samples with direct amplification and the unconcentrated samples with DNA extraction and amplification.

<p>This 1:1 plot shows amplification results of the field-concentrated samples with direct amplification as compared to the results of 1 L unconcentrated samples following DNA extraction. Points along the y-axis only amplified with the field-concentrated samples and direct amplification while those along the x-axis only amplified with the unconcentrated method. Points in the center correspond to positive detections using both methods.</p

List of LAMP primers used in this study.

<p>List of LAMP primers used in this study.</p

Results obtained for different sample types including: <i>Dreissena polymorpha</i> tissues, <i>Dreissena bugensis</i> tissues, <i>D</i>. <i>polymorpha</i> veligers, 1000X concentrated water, and un-concentrated water.

<p>Information for each sample includes the location, the month and year of sample collection, sample processing, primers used, estimated target per reaction, and measured TTP.</p

Direct amplification results for sample collection strategies including 1000X concentration (20 L hand-filtered to 20 mL) and un-concentrated water, at high initial population abundances (circles; open for concentrated and closed for un-concentrated) and low initial population abundances (triangles; open for concentrated and closed for un-concentrated).

<p>At high abundance, no change in TTP was observed between 1000X concentration and water-only. At low abundance, positive results were observed only after 1000X concentration.</p

Results from direct amplification of environmental samples.

<p>Mass estimates for <i>D</i>. <i>polymorpha</i> CO1 (blue circles), <i>D</i>. <i>bugensis</i> CO1 (red triangles), and <i>Dreissena</i> sp. 18S rRNA (black squares). Larger shapes correspond to a high concentration tissue detected.</p

Isothermal amplification of environmental DNA (eDNA) for direct field-based monitoring and laboratory confirmation of <i>Dreissena</i> sp.

<div><p>Loop-mediated isothermal amplification (LAMP) of aquatic invasive species environmental DNA (AIS eDNA) was used for rapid, sensitive, and specific detection of <i>Dreissena</i> sp. relevant to the Great Lakes (USA) basin. The method was validated for two uses including i) direct amplification of eDNA using a hand filtration system and ii) confirmation of the results after DNA extraction using a conventional thermal cycler run at isothermal temperatures. Direct amplification eliminated the need for DNA extraction and purification and allowed detection of target invasive species in grab or concentrated surface water samples, containing both free DNA as well as larger cells and particulates, such as veligers, eggs, or seeds. The direct amplification method validation was conducted using <i>Dreissena polymorpha</i> and <i>Dreissena bugensis</i> and uses up to 1 L grab water samples for high target abundance (e.g., greater than 10 veligers (larval mussels) per L for <i>Dreissena</i> sp.) or 20 L samples concentrated through 35 μm nylon screens for low target abundance, at less than 10 veligers per liter water. Surface water concentrate samples were collected over a period of three years, mostly from inland lakes in Michigan with the help of a network of volunteers. Field samples collected from 318 surface water locations included i) filtered concentrate for direct amplification validation and ii) 1 L grab water sample for eDNA extraction and confirmation. Though the extraction-based protocol was more sensitive (resulting in more positive detections than direct amplification), direct amplification could be used for rapid screening, allowing for quicker action times. For samples collected between May and August, results of eDNA direct amplification were consistent with known presence/absence of selected invasive species. A cross-platform smartphone application was also developed to disseminate the analyzed results to volunteers. Field tests of the direct amplification protocol using a portable device (Gene-Z) showed the method could be used in the field to obtain results within one hr (from sample to result). Overall, the direct amplification has the potential to simplify the eDNA-based monitoring of multiple aquatic invasive species. Additional studies are warranted to establish quantitative correlation between eDNA copy number, veliger, biomass or organismal abundance in the field.</p></div