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Development of a Real-time PCR Method for Early Detection of Aquatic Invasive in Pennsylvania Watersheds

The widespread occurrence of aquatic invasive species is a worldwide issue threatening the environment and impacting the intended use of water. Conventional methods for the identification of aquatic invasive species are laborious and time-consuming, preventing the timely application of eradication measures. On the contrary, molecular biology methods based on the detection of specific DNA sequences are fast and sensitive and have raised increasing interest for monitoring invasive species. All aquatic organisms release in water small amounts of DNA, which constitutes a signature unique to the species. Analyzing environmental DNA in water for the identification of such unique markers is a powerful strategy for detecting low density invasive species in a water body.

The objective of the proposed research was to develop and optimize an innovative method (based on real-time PCR) for the early detection of three aquatic invasive plants, Myriophyllum spicatum, Trapa natans, and Hydrilla verticillata, and the invasive alga, Didymosphenia geminata, which all constitute a threat for Pennsylvania watersheds. The proposed research involved three specific objectives:
1. To design assays based on real-time PCR for the detection of the target invasive species.
2. To validate the assays and optimize experimental protocols.
3. To test the applicability and robustness of the assays in the field.

Using resources from genomic databases and sequence alignment tools, we designed primers and fluorescent probes specific to the target species, T. natans, M. spicatum, H. verticillata, and D. geminata. The primer and probes allowed the detection of DNA signatures from the target invasive species using real-time PCR. The specificity of the primers and probes was successfully tested against a suite of relative plant species. The quality control and validation of the method provided overall satisfactory results: near-perfect linearity was observed with all standard curves, the dynamic range of the detection method expanded over at least five orders of magnitude, and the amplification efficiency was within the adequate range, except for one of the assay (M. spicatum), which will require additional optimization. Three field studies provided results consistent with the expectations. When monitoring the three target invasive plant species, T. natans, H. verticillata, and M. spicatum, specific DNA signatures were detected in water samples miles away from the recognized contamination point. The signal intensities were generally several orders of magnitude higher than the signal intensities obtained with negative control samples. When monitoring D. geminata, the specific DNA signature was detected in water samples hundred yards downstream a location where the species was historically detected, even though the presence of the invasive alga was not visible at the time of sampling.

The innovative aspect of the developed assays relies on the possibility to detect the target species based on the presence of DNA signatures in the water, i.e., without the need to physically observe or collect specimens. The developed method is expected to allow fast screening of a large number of water bodies with limited personal investment, as it does not require time-consuming microscopic identification.

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