Abstract
Loop-mediated isothermal amplification (LAMP) of aquatic invasive species environmental DNA (AIS eDNA) was used for rapid, sensitive, and specific detection of Dreissena 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 Dreissena polymorpha and Dreissena bugensis and uses up to 1 L grab water samples for high target abundance (e.g., greater than 10 veligers (larval mussels) per L for Dreissena 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.
Highlights
The use of environmental DNA for aquatic invasive species (AIS) detection has the potential to increase the likelihood of early detection [1] and enhance the probability of successful eradication [2]
The results obtained in this study through the collection and analysis of 318 samples supports that direct amplification may be useful for field monitoring of aquatic invasive species
While this is not the first study to analyze large numbers of samples for environmental DNA (eDNA) from aquatic invasive species [52] including Dreissena sp. [53], it is the first of its kind to analyze large numbers of samples for Dreissena sp. using loop-mediated isothermal amplification (LAMP). This highlights the advantages of a direct amplification-based eDNA approach, in that large numbers of samples are analyzed for dozens of species in a short time
Summary
The use of environmental DNA (eDNA) for aquatic invasive species (AIS) detection has the potential to increase the likelihood of early detection [1] and enhance the probability of successful eradication [2]. Simplifying the analytical approach and decreasing the time-to-result is a key first step in developing rapid, field-deployable nucleic acid- based eDNA detection methods. I.e., amplification without DNA extraction or purification, satisfies both these attributes. Elimination of DNA extraction and purification steps simplifies the process and may avoid the need for sample transport [3,4]. For detection of invasive species at very low abundance, sample concentration is often useful and necessary. Sample concentration may lead to simultaneous concentration of substrates inhibitory to Taq polymerases used in polymerase chain reaction (PCR)-based eDNA assays [5]
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