I.S. and C.D.S. contributed to study conception and design. I.S. secured funding for assay development, testing, and field survey work. I.S., K.-A.B., and S.B. conducted field surveys in Jamaica; I.S. and J.L.W. oversaw collection of positive and negative control samples in Oklahoma. Assay design was undertaken by E.D.H. DNA extractions, in vitro assay tests, and assessment of assay performance was overseen by S.N.S. Data analysis and summary of qPCR screening results was performed by S.N.S. and C.D.S., with contributions from J.L.W. The first draft of the manuscript was written by S.N.S., with contributions and reviews by C.D.S., I.S., J.L.W., E.D.H., K.-A.B., and S.B. All authors read and approved the final manuscript.We describe the development and validation of a qPCR assay to detect Poecilia reticulata, a highly invasive species of freshwater fish invasive to the Caribbean islands, through environmental DNA (eDNA) sampling. Originating from Trinidad, this species is invasive and detrimental to countless native tropical fish communities. A qPCR assay, consisting of a set of primers and a fluorescent probe, amplifying a 214 base pair target region of the mitochondrial Cytochrome B gene was designed for P. reticulata from existing DNA sequence data. The assay was assessed for target specificity, with no evidence of amplification in closely related or sympatrically distributed non-target species. In vitro tests indicate that the assay consistently detects P. reticulata down to concentrations of 2.0 × 10−5 ng/μl. The developed assay provides a new, practical tool for monitoring freshwater habitats throughout the Caribbean, allowing for early and rapid detection of invasive fish species of conservation concern.This research was supported by the University of Oklahoma, the Sam Noble Oklahoma Museum of Natural History, and the University of the West Indies, Mona Campus. Research was conducted in accordance with field permits in Jamaica (NEPA#18/27) and the University of Oklahoma Institutional Animal Care and Use Committee (IACUC) protocols R17-031 and R19-006. The authors thank M. Gordon, H. Bennett, E. Bennett and S.-M. Bennett-Monroe for field and laboratory assistance. Funding support for this work was provided by the University of Oklahoma and the Sam Noble Oklahoma Museum of Natural History to C.D.S and I.S.Ye
