Abstract
Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated on the specificity and sensitivity of the PCR assay, careful validation of each eDNA qPCR assay in development must be performed both under controlled laboratory conditions and when challenged with field-derived eDNA samples. Such a step-wise approach forms the basis for incorporation of innovative qPCR design features that strengthen the implementation and interpretation of the eDNA assay. This includes empirical determination that the qPCR assay is refractory to the presence of human DNA and the use of a tripartite assay approach comprised of 1) a primer set targeting plant chloroplast that evaluates the presence of amplifiable DNA from field samples to increase confidence in a negative result, 2) an animal group primer set to increase confidence in the assay result, and 3) a species-specific primer set to assess presence of DNA from the target species. To demonstrate this methodology, we generated eDNA assays specific for the North American bullfrog (Lithobates (Rana) catesbeiana) and the Rocky Mountain tailed frog (Ascaphus montanus) and characterized each with respect to detection sensitivity and specificity with demonstrated performance in a field survey scenario. The qPCR design features presented herein address specific challenges of eDNA assays thereby increasing their interpretative power.
Highlights
In the realm of environmental science, an understanding that genetic material can be harvested from ecosystem biota has been known for decades [1]
Development of a polymerase chain reaction (PCR)-based assay that serves to detect the presence of a single animal species or a select group of related animals from a complex environmental DNA source is no small feat
In addition to appropriate sample collection methodology, an ‘informed design’ must be performed for the molecular assay whereby known evolutionarily closely related species, members of the same animal family that are situated in the geographic survey region, and similar human sequences to the target gene are all factored into the primer design process in an attempt to maximize eDNA assay specificity towards the target species and/or more broadly towards a given family or genera
Summary
In the realm of environmental science, an understanding that genetic material can be harvested from ecosystem biota has been known for decades [1]. The presence of both the DNA amplification primer pair and a hydrolysis probe in assay development serves to enhance target specificity by allowing for increased placement of cross-species base-pair mismatches that minimize false positive detection of related members of the animal family or genus [19].
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