Quantitative PCR multiplexes for simultaneous multispecies detection of Asian carp eDNA Kristyne M. Wozney ⁎, Chris C. Wilson Aquatic Research and Monitoring Section, Ontario Ministry of Natural Resources and Forestry, Trent University, 2140 East Bank Drive, Peterborough, Ontario K9L 0G2, Canada abstract article info Article history: Received 6 December 2016 Accepted 23 May 2017 Available online xxxx Environmental DNA (eDNA) is increasingly being used as a surveillance tool for detection of aquatic invasive spe- cies. In particular, this method is being applied by many jurisdictions surrounding the Great Lakes to monitor for the potential presence of Asian carp. We developed two multiplex quantitative PCR (qPCR) reactions for the si- multaneous testing for eDNA from the four species of Asian carp that have been identified as potential invaders into the Great Lakes [bighead carp (Hypophthalmichthys nobilis), silver carp (Hypophthalmichthys molitrix), black carp (Mylopharyngodon piceus), and grass carp (Ctenopharyngodon idella)], along with internal controls to test for false positive results due to sample contamination. The assays are highly sensitive and have the ability to detect trace amounts of carp DNA in laboratory controls. The use of a multiplex design allows for simultaneous testing for numerous species, reducing processing and handling times, lowering costs and reducing the amount of DNA extract required for testing. The multiplex assays described in this study may be readily applied to Asian carp monitoring programs, and are currently being used to support surveillance efforts in the Canadian waters of Lake Erie, Lake Huron and Lake Ontario for all four species of Asian carp. © 2017 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved. Keywords: eDNA Asian carp Bighead carp (Hypophthalmichthys nobilis) Silver carp (Hypophthalmichthys molitrix) Grass carp (Ctenopharyngodon idella) Black carp (Mylopharyngodon piceus) Introduction Early detection of aquatic invasive species (AIS) is essential for de- veloping and implementing rapid response strategies to prevent their establishment (Amberg et al., 2015; Anderson, 2005; Smart et al., 2015; Vander Zanden et al., 2010). In aquatic environments, capture of low density species with traditional field sampling can be difficult; as a result, molecular methods for detection are becoming more widely used for species detection (Boothroyd et al., 2016; Thomsen et al., 2011). In particular, environmental DNA (eDNA) detection provides a sensitive method for targeted early detection of aquatic invasive species (Darling and Blum, 2007; Goldberg et al., 2013; Jerde et al., 2011; Smart et al., 2015). Detection tools for large scale surveillance efforts should be sensitive, reliable, cost-effective and have a short turn-around time. Pre- vious studies suggest that eDNA has the potential to identify species oc- curring at low abundance, enabling population monitoring with less cost and effort than traditional sampling methods (Dejean et al., 2011; Ficetola et al., 2008). The four species of Asian carp that have been identified as high-risk potential invasives for the Great Lakes [bighead carp (Hypophthalmichthys nobilis), silver carp (H. molitrix), grass carp (Cteno- pharyngodon idella), and black carp (Mylopharyngodon piceus)] were brought to North America predominantly for use in aquaculture in the southern United States and have subsequently escaped into the wild (Cudmore and Mandrak, 2011; Nico et al., 2005). Since their introduc- tion, populations of bighead and silver carp have steadily increased and now comprise a major part of the biomass in the Mississippi River basin (ACRCC, 2012; Cudmore and Mandrak, 2011; Herborg et al., 2007; Kolar et al., 2005). Adult grass carp have been captured in all of the Great Lakes with the exception of Lake Superior, and there is strong evidence of grass carp spawning in a tributary to Lake Erie (Chapman et al., 2013; Embke et al., 2016). While not as abundant as the other Asian carps, black carp are also expanding their range with reports of black carp captures increasing dramatically over the past decade (Kolar et al., 2007; Nico et al., 2005). The probability of establishment for each of the above species is con- sidered to be high and could have significant ecological and economic impacts (Cudmore and Mandrak, 2011; Cudmore et al., 2011; Kocovsky et al., 2012; Nico et al., 2005; Vander Zanden et al., 2010). In addition, risk assessment models suggest there is a significant risk of es- tablishment from low numbers of founding individuals (Cuddington et al., 2014; Cudmore et al., 2011). It is essential to establish sensitive and reliable detection methods which would facilitate rapid response ac- tions in the early stages of colonization (Anderson, 2005; Vander Zanden et al., 2010). Immediate action may then prevent establishment and subsequent expansion in the Great Lakes and connected inland wa- ters. For Asian carp it has been shown that eDNA has a greater detection sensitivity than traditional netting and electrofishing (Jerde et al., 2011) and many jurisdictions bordering the Great Lakes have adopted this Journal of Great Lakes Research xxx (2017) xxx–xxx ⁎ Corresponding author. E-mail address: kristyne.wozney@ontario.ca (K.M. Wozney). JGLR-01223; No. of pages: 6; 4C: http://dx.doi.org/10.1016/j.jglr.2017.05.001 0380-1330/© 2017 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Great Lakes Research journal homepage: www.elsevier.com/locate/jglr Please cite this article as: Wozney, K.M., Wilson, C.C., Quantitative PCR multiplexes for simultaneous multispecies detection of Asian carp eDNA, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.05.001