Quantitative PCR multiplexes for simultaneous multispecies detection of Asian carp eDNA

Environmental DNA (eDNA) is increasingly being used as a surveillance tool for detection of aquatic invasive species. 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 simultaneous 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.