![]() ![]() Field conditions differ from laboratory conditions because in the field PCR inhibitors are more likely to be present in samples. Again, the assay should be re-assessed when used in a new location. Assay specificity may change when applied to a different geographic region, because the assay is being applied to samples from a new biotic community that may include non-target species that the assay has not been tested against, and genetic variation in the target species may occur. Even assays well-characterized in the literature should be tested and optimized when adopted by a new laboratory or when using different reagents (e.g., master-mix solution) 5, 9. Therefore, this protocol should be revisited when applying an assay under new conditions. This can occur under different laboratory conditions (i.e., different reagents, users, machines, etc.). Because there are many natural sources of variation in eDNA, studies must limit controllable sources of variation as much as possible, including fully optimizing and characterizing the eDNA assay 3.Ĭonditions that directly affect an assay’s specificity or sensitivity will change the assay’s performance. An understanding of assay sensitivity and selectivity will help inform the sampling effort needed to detect rare species. Assays that cross-react with non-target species DNA could lead to false positive detections, while assays with poor sensitivity may fail to detect the target species DNA even when it is present in the sample (false negatives). Optimization and reporting of assay performance aids in study design and interpretation of eDNA survey results. Reporting quantitative measures of assay performance has been previously largely overlooked, but recently standards to improve transparency in assay development are emerging 3, 4, 5, 6, 7, 8. An assay’s reliability in detecting only the DNA of a target species (i.e., specificity) and detection of low quantities of target DNA (i.e., sensitivity) may vary considerably due to differences in how the assay was designed, selected, optimized and tested. It is vital to understand that published assays for eDNA samples are not always equal in performance. Mitochondrial DNA sequences are generally targeted in eDNA assays because the mitochondrial genome is present in thousands of copies per cell, but assays for nuclear DNA or RNA sequences are also possible. Within the relatively new field of eDNA research, use of these assays with a standard curve for quantification of copies of target DNA per volume or weight of eDNA sample has now become routine practice. For three decades, quantitative or real time PCR (qPCR/rtPCR) has been used in numerous fields for the sequence-specific detection and quantification of nucleic acids 1, 2. Researchers and managers are increasingly becoming interested in the use of environmental DNA assays for species detection. We demonstrate this process with our assay designed for populations of the mucket ( Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. ![]() ![]() In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Using probe-based qPCR offers greater specificity than is possible with primers alone. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. ![]()
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