Environmental DNA Marker Workshop
BY NICHOLAS BERNDT AND EMY MONROE, WHITNEY GENETICS LAB
Researchers, biologists, and technicians from all areas of invasive carp work and water resource management visited the USGS Upper Midwest Environmental Science Center (UMESC) environmental DNA (eDNA) marker workshop. This workshop showcased cutting edge, real-time Polymerase Chain Reaction (PCR) techniques, new invasive carp marker development, and recommendations for new markers to be used to analyze 2014 eDNA samples from across the Great Lakes and Mississippi River systems. The event was co-hosted by the USFWS Whitney Genetics Laboratory (WGL), the USGS Upper Midwest Environmental Sciences Center (UMESC), and the US Army Corps of Engineers Research and Development Center (ERDC). This workshop was the result of the Marker Validation Study which was co-lead by Emy Monroe of WGL, Chris Rees of UMESC, and Rick Lance of ERDC, and the results presented were much anticipated by all in attendance.
New real-time PCR markers were developed by ERDC and UMESC where they underwent rigorous testing and optimization before becoming viable candidates for use in the monitoring program. During development, these new markers were found to have greater sensitivity then cPCR markers, were faster to process, and could even look for multiple species in one reaction. Even though these benefits were demonstrated in the development labs, in order to be implemented into the monitoring program, the markers required validation in a three-lab round-robin, double-blind study to demonstrate consistency among technicians and instrumentation and to demonstrate sensitivity and function in various carp-positive and carp-negative water sources from different locations. Thirteen new markers were developed and passed the validation criteria and the real-time markers outperformed cPCR markers for silver and bighead carp detection. They had highly reproducible results, were more sensitive and accurate than cPCR markers, and cut down on the effects of inhibition in eDNA PCR reactions. Of the 13 markers passing validation, six real-time PCR markers were proposed for use in the 2014 QAPP for the monitoring season. The new real-time markers can detect and confirm DNA presence, which saves time and money, and reduces the risk of contamination in the lab.
Once the workshop was presented, resource managers were given time to discuss the new markers, ask questions, and finally approve use of the new markers for the 2014 season. The following week, staff at WGL began training and transitioning staff that had not been part of the Marker Validation study on all the ins and outs of the real-time PCR technique. Incidentally, those staff were not part of Marker Validation, because they were busy extracting the thousands of samples (multiplied into thousands more filters) in freezers at WGL. With the switch to more sensitive and accurate real-time markers comes the need for extra vigilance in the lab and even more refined bench techniques. Similar to the transition plan executed when WGL assumed eDNA processing from ERDC in 2013, a transition plan was drafted and executed in-house at WGL. All lab personnel passed the transition look forward to utilizing the new markers. With an updated suite of genetic markers to work with, WGL will have the more refined techniques for the early detection of invasive carp, which in turn provides the best available information for our water resource managers.