Comparison of the efficiency of three methods of isolating fish DNA from environmental samples

Abstract

The Lake Ecosystem Restoration New Zealand (LERNZ) project was initiated to restore indigenous biodiversity in lakes in part by developing new pest fish management and control technologies. One objective of the management of pest fish was to develop genetic based assays to detect pest fish from environmental samples such as water, and to assess the feasibility of using these methods in the field. This report compares three methods (the Piscitron II, Piscitron III and a pH adjusted method) of isolating DNA for two species of exotic fish from water samples. We compared DNA isolation efficiency using newly developed qPCR assays for koi carp, Cyprinus carpio and goldfish, Carassius auratus , and present the primer and hydrolysis probe sequences for the assays, and assay thermal conditions. Minimum levels of detectable fish biomass are reported for koi carp water collected from a 6800 L tank at ambient temperature containing a 1.6 kg koi carp. The koi carp and goldfish assays were also tested on Waikato River water collected next to Hammond Park, Hamilton, New Zealand, an area of river known to contain koi carp and goldfish. We found that the Piscitron II and Piscitron III methods, which filtered water through glass fibre filters or glass wool aquarium filters, respectively, performed equally well in terms of DNA extraction but considerably better than the pH adjusted method. Minimum detectable amounts of koi carp DNA were calculated by producing serial dilutions of the tank water containing 2.35* 10⁻⁴ kg of koi carp/L. Because fish densities tend to be reported as kg/m² of lake surface, we converted our minimum detectable amount from a weight per volume to a weight per area by assuming an average lake depth of 1.5 m (a typical Waikato lake depth) which resulted in a minimum detectable amount of 0.35 kg carp/m² of water surface area. We also produced serial dilutions of DNA extracted from the koi carp tank and the minimum detectable amount of koi carp biomass was much lower at 0.0035 kg/m² which is less than the range of carp densities (0.005-0.0768 kg/m²) reported for Waikato lakes and rivers.