Implementation of Novel Design Features for qPCR-Based eDNA Assessment.
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.
Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055, STN CSC, Victoria, British Columbia, V8W 2Y2, Canada.
Nik
N
Veldhoen
Hemmera Envirochem Inc., 303-1221 Broad Street, Victoria, British Columbia, V8W 2A4, Canada.
Jared
J
Hobbs
Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055, STN CSC, Victoria, British Columbia, V8W 2Y2, Canada.
Georgios
G
Ikonomou
Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055, STN CSC, Victoria, British Columbia, V8W 2Y2, Canada.
Michael
M
Hii
Department of Mathematics and Statistics, 3800 Finnerty Road, University of Victoria, Victoria, British Columbia, V8P 5C2, Canada.
Mary
M
Lesperance
Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055, STN CSC, Victoria, British Columbia, V8W 2Y2, Canada.
Caren C
CC
Helbing
D000818:Animals; D001001:Anura / Q000235:genetics; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D017753:Ecosystem; D004777:Environment; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D017931:DNA Primers; D004247:DNA