title: Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.
abstract: Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.
authors: †Institute for Sustainable Sciences and Development, Hiroshima University, Higashi-Hiroshima 739-8530, Japan.
authors: Hideyuki
authors: H
authors: Doi
authors: ‡Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan.
authors: Teruhiko
authors: T
authors: Takahara
authors: §Graduate School of Human Development and Environment, Kobe University, Kobe 657-8501, Japan.
authors: Toshifumi
authors: T
authors: Minamoto
authors: †Institute for Sustainable Sciences and Development, Hiroshima University, Higashi-Hiroshima 739-8530, Japan.
authors: Saeko
authors: S
authors: Matsuhashi
authors: ∥Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi 584-0066, Japan.
authors: Kimiko
authors: K
authors: Uchii
authors: ⊥Department of Environmental Solution Technology, Faculty of Science and Technology, Ryukoku University, Otsu 520-2194, Japan.
authors: Hiroki
authors: H
authors: Yamanaka
mesh_terms: D000818:Animals; D004247:DNA / Q000235:genetics*; D017753:Ecosystem; D004777:Environment; D005453:Fluorescence; D058865:Introduced Species; D057230:Limit of Detection; D010473:Perciformes / Q000235:genetics*; D061005:Ponds; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014867:Water / Q000032:analysis
chemical_list: D014867:Water; D004247:DNA