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Droplet digital PCR outperforms real-time PCR in the detection of environmental DNA from an invasive fish species Hideyuki Doi, Teruhiko Takahara, Toshifumi Minamoto, Saeko Matsuhashi, Kimiko Uchii, and Hiroki Yamanaka Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00253 • Publication Date (Web): 07 Apr 2015 Downloaded from http://pubs.acs.org on April 12, 2015

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Environmental Science & Technology

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Droplet digital PCR outperforms real-time PCR in the detection

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of environmental DNA from an invasive fish species

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Hideyuki Doi ‡1 *, Teruhiko Takahara‡2, Toshifumi Minamoto3, Saeko Matsuhashi1, Kimiko

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Uchii4, and Hiroki Yamanaka5

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739-8530 Japan 2 Graduate School of Integrated Arts and Sciences, Hiroshima University, 739-

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8530 Higashi-Hiroshima, Japan 3 Graduate School of Human Development and Environment,

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Kobe University, Kobe 657-0013 Japan 4 Faculty of Pharmacy, Osaka Ohtani University,

Institute for Sustainable Sciences and Development, Hiroshima University, Higashi-Hiroshima,

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Tondabayashi 584-0066 Japan 5 Department of Environmental Solution Technology, Faculty of

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Science and Technology, Ryukoku University, Otsu 520-2194 Japan

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*Corresponding author: Hideyuki Doi

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Institute for Sustainable Sciences and Development, Hiroshima University,

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Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8530 Japan

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e-mail: [email protected]

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Tel: +81-82-424-5732

Fax: +81-82-424-5732

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ACS Paragon Plus Environment

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ABSTRACT

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Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems.

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Most of these studies use real-time PCR to detect eDNA in water; however, PCR amplification is often

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inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is

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partitioned into thousands of nano-liter droplets, and PCR inhibition may be reduced by the detection of

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the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In

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addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We

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compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the

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detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that

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ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the

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PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by

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spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that

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ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to

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the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time

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PCR methods for detecting eDNA to document species distributions in natural habitats, especially in

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habitats with high concentrations of PCR inhibitors.

37 38


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Introduction

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Documenting species distributions is fundamental to ecosystem management and is essential 1-3

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for managing and conserving rare and endemic species,

as well as for controlling invasive

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species. Recently, environmental DNA (eDNA) analyses have been applied to detect the

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presence of species in aquatic and terrestrial ecosystems.4, 5 eDNA surveys have great potential

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to contribute to species distribution data and reduce survey costs. eDNA methods have been

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applied for the detection of many types of animals, especially in aquatic ecosystems; these

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include common, 6-13 invasive, 14-19 and endangered species20-23 in various ecosystems, including

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ponds and lakes, 10, 11,15,17,18 rivers and streams, 6, 7, 9, 12, 16, 19, 23 and oceans. 14

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Most of these eDNA studies have used PCR techniques to detect eDNA originating from the

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target species, thereby assessing the presence or absence of the species.6-9 eDNA detection was

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first used in the field to assess the status of the bullfrog Rana catesbeiana (= Lithobates

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catesbeianus) using electrophoresis on PCR products. 6 Recent eDNA studies generally use real-

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time PCR platforms.9, 10, 20, 21 For example, Takahara et al. used real-time PCR to detect the

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eDNA of invasive bluegill sunfish (Lepomis macrochirus) in ponds in Japan, obtaining better

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detection rates than traditional observation methods.

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crayfish, Procambarus clarkii, and New Zealand mud snails, Potamopyrgus antipodarum, have

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been assessed using real-time PCR eDNA detection.16, 17

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Also, the distributions of invasive

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However, it is known that eDNA detection rates can be limited by very-low DNA sample

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concentrations and the presence of substances which inhibit PCR.11-13 Increasing eDNA

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concentrations in field samples by concentrating large volumes of water may also increase the

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concentrations of inhibitory substances in the form of macromolecules present in the natural


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environment. For example, humic and tannic acids, polymers, and many unknown materials are

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commonly present in crude DNA extracts from soils and tissues, and even small quantities may

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be sufficient to inhibit Taq polymerase.24,25,26 To extract and purify eDNA samples, thus

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reducing concentrations of such PCR inhibitors, eDNA studies generally use DNA purification

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kits, but these do not eliminate all macromolecules. Thus, we need to develop eDNA detection

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methods with high sensitivities and tolerances to inhibitory substances.

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Recently, droplet digital PCR (ddPCR), also known as a “third-generation PCR,” has been

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developed.24 ddPCR is an emerging DNA detection method which generates thousands of nano-

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liter droplets, some of which ideally contain only one or few copies of the target DNA. The PCR

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reaction occurs in each droplet, and end-point PCR amplification is detected by the fluorescence

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intensity of PCR probes.27 To date, ddPCR has been used as an alternative of real-time PCR to

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quantify DNA concentrations.28-33 Recently, ddPCR has been applied to quantify fish eDNA

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concentrations in mesocosm experiments.34-35 In ddPCR, PCR inhibitory substances should have

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little effect on DNA quantification, because the end-point PCR amplification in each droplet can

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be detected independent of the amplification efficiency.27,36 Thus, we may expect ddPCR to be

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more suitable for the measurement of eDNA in field samples than real-time PCR. However, the

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performance of ddPCR in detecting eDNA in field samples has never been evaluated.

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In many real-time PCR eDNA studies, TaqMan Gene Expression Master Mix (GEMM, Life

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Technologies) has been used because of its highly sensitive DNA detection. Recently, TaqMan

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Environmental Master Mix 2.0 (EMM, Life Technologies) has been used for eDNA

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detection14,17,19. EMM is tolerant to high concentrations of PCR inhibitors occurring in field

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water samples, such as humic acid, and may improve eDNA detection. While a previous study11

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showed that a real-time PCR with EMM detected target eDNA from several samples in which a


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real-time PCR with GEMM detected no amplification, little is known so far about the relative

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performance of the two for eDNA detection rate. In this study, we compared the accuracies of 1)

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ddPCR, 2) real-time PCR with GEMM, and 3) real-time PCR with EMM in detecting eDNA of

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invasive bluegill sunfish in field samples obtained from ponds, as studied by Takahara et al.15

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We evaluated the differences in eDNA detection rates and limits of detection (LOD) using the

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three PCR methods.

91 92

Materials and Methods

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eDNA Sampling

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We examined the presence of invasive bluegill sunfish (L. macrochirus) populations in ponds,

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which were previously surveyed for eDNA by Takahara et al.15. These Japanese populations

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were derived from a small number of individuals from a population in the Mississippi River,

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USA.37 We used eDNA samples from 25 ponds which had been previously collected by

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Takahara et al.15. In 19 out of the 25 ponds, bluegill eDNA had previously been detected in at

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least in one of eight replicates in our previous study using real-time PCR with GEMM.15 The

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other 6 ponds were randomly selected from the ponds in which eDNA was not detected and

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bluegill was not found visually from the shore with walking of the whole shoreline for 10–20

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min. Briefly, 1 L of water was collected from the surface of each pond from October to

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December 2011. The water samples were stored in DNA-free bottles (Nalgene®) and

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immediately transported in a cooling box on ice to the laboratory and stored at –30°C until

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processed. After thawing, the water samples were filtered through a 3.0 µm membrane filter

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(cellulose acetate, C300A142C; Advantec, Saijo, Japan). The filter discs were then placed in


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autoclaved bottles and soaked in 10 mL autoclaved ultrapure water and agitated on a rotary

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shaker at the maximum speed (250 rpm) for 10 min. Suspended matter in the bottles was

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decanted into a centrifugal filter unit S10Y30 (30-kDa cutoff, 540642; Millipore, Billerica, MA)

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and concentrated to 400 µL by spinning at 5000 × g. for 15 min. Then, eDNA was extracted

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using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) in a final volume of 100 µL. As

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blanks, DNA-free pure water (1 L) were treated in the same manner as the samples during

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filtration, eDNA extraction and PCR procedures, and bluegill eDNA was not detected in these in

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subsequent ddPCR and real-time PCR assays. Full details of the methods for eDNA sampling

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and(?) extraction have been described previously by Takahara et al.15

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Real-time PCR assays

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Real-time PCR for bluegill mitochondrial cytochrome b gene fragments was performed as

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described by Takahara et al.15 eDNA was quantified using real-time TaqMan® PCR with a

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StepOnePlus™ Real-Time PCR system (Life Technologies, Carlsbad, CA, USA). The

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mitochondrial cytochrome b gene fragments (100 bp) were amplified and quantified with the

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following

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GCCTAGCAACCCAGATTTTAACA-3′),

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ACGTCCCGGCAGATGTGT-3′),

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CGACATCGCAACTGCCTTCTCTTCAGT-TAMRA-3′). 15 The specificity of the primers and

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probe was tested with extracted DNA samples from other sunfish species present in Japan

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(Micropterus salmoides and M. dolomieu) using real-time PCR, which generated no

primers

and

TaqMan

probe:

Bluegill_CytB_F

Bluegill_CytB_R

and

Bluegill_CytB_probe


(5′-

(5′-

(5′-FAM-

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amplification signals. In addition, we directly sequenced the PCR amplicons to confirm the

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specificity of the primer set. To confirm the specificity of the primer set in the field, real-time

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PCR amplicons of all eDNA samples that were positive for the bluegill DNA were directly

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sequenced after treatment with ExoSAP-IT (USB Corporation, Cleveland, OH, USA). Sequences

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were determined by a commercial sequencing service (Takara Bio, Tokyo, Japan). All sequences

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from each real-time PCR amplicon were confirmed as being from bluegill.

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We used two master mix types (TaqMan Gene Expression Master Mix, GEMM, and TaqMan

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Environmental Master Mix 2.0, EMM, Life Technologies) for real-time PCR. Each TaqMan

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reaction contained 900 nM of each primer and 125 nM of TaqMan probe in 1× PCR master mix

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kit (GEMM or EMM), along with 2 µL of DNA solution. Takahara et al.11 found that 2 µL of

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DNA solution was optimal to detect fish eDNA using real-time PCR with minimum PCR

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inhibitory effects for the pond samples. Eight replicates of each PCR reaction were performed in

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a thermocycler (2 min at 50°C, 10 min at 95°C, and 55 cycles of 15 s at 95°C and 60 s at 60°C)

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in a StepOnePlus Real Time PCR system (Life Technologies). We used 55 PCR cycles following

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the standard protocol used in eDNA studies to increase eDNA detectability.11, 14, 15 Eight wells

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were used as no-template negative controls for all real-time PCR assays; no amplification signal

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was observed in these wells or the blank samples. To avoid contamination, we performed

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the PCR set-up, including preparation and addition of the standards, in a separate room from that

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where PCR cycling occurred.

148 149

ddPCR assays

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We performed ddPCR analysis using the same DNA solutions and the same primers and probe

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used for real-time PCR analysis. Each ddPCR reaction mixture (total 20 µL) contained 2 µL of


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DNA solution, 900 nM of each primer, and 125 nM TaqMan probe in a 1× Bio-Rad Supermix kit

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(Bio-Rad, Hercules, CA, USA), which was mixed with Bio-Rad droplet generator oil and

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partitioned into nano-litter droplets using a Bio-Rad QX-100 droplet generator (Bio-Rad).

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Droplets of each sample were separately applied to each well of a 96-well PCR plate. PCR was

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performed with the 96-well plate sealed with pierceable sealing foil using a GeneAmp 9700

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thermocycler (Applied Biosystems, Grand Island, NY, USA). PCR conditions were 10 min at

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95°C, 45 cycles of denaturation for 30 s at 95°C and extension for 60 s at 60°C with ramp rate of

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2.5°C s-1, followed by 10 min at 95°C and a hold at 4°C. Based on the results of a preliminary

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experiment (Appendix S1) and the maximum Ct values of real-time PCR (40.8) in this and our

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previous study,15 we decided to measure eDNA for all samples using PCR with 45 cycles. After

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amplification, the PCR plate was transferred to a QX-100 droplet reader (Bio-Rad). The TaqMan

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fluorescence of each droplet in each well was measured. Eight wells were used as no-template

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negative controls for all ddPCR assays; droplets from the negative-control samples had

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fluorescence (RFU) less than 800 after PCR amplification. We used Bio-Rad’s QuantaSoft

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software version 1.3.2.0 to quantify the detection of target DNA. The threshold for a positive

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signal was set at 1100 RFU, as determined by the negative control in LOD analysis, described

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below. Any droplet with fluorescence above this threshold was counted as positive for bluegill

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eDNA (Fig. 1, Fig. S1). The mean volume of droplet will be about 1 pL with 20-µL PCR

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reaction. The mean detected droplets and analysis variance by ddPCR were 10,081 (± 358, 95%

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CI) and 35.9 for all ddPCR analysis, respectively. We included all essential (E) information in

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dMIQE required37 (Appendix).

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Testing limit of detection (LOD)


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To compare the minimum number of target DNA copies that could be detected among PCR

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types (ddPCR, real-time PCR with GEMM, and real-time PCR with EMM), we performed two

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limit of detection (LOD) analysis for each method. 1) DNA dilution experiment; we used the

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following dilution series of bluegill DNA standard: 0, 1, 2, 3, and 10 copies per 2-µL PCR

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template. The bluegill DNA used for LOD analysis was cloned with pGEM-T Easy Vector

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(Promega, Tokyo, Japan), which concentration was quantified using a NanoDrop 1000

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spectrophotometer (Thermo Fisher Scientific, Waltham, MA USA)15. For each PCR method, we

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performed eight replicated LOD analyses for each number of DNA copies in the dilution series.

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2) DNA spiking experiment; we mixed 10 pond samples (26 µL each), from which bluegill

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eDNA were not detected, to prepare 260 µL of mixed DNA solution. The mixed DNA solution

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was spiked with the bluegill DNA at concentrations of 0, 1, 2, 3, and 10 copies µL–1. Real-time

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PCR and ddPCR were performed with 2 µL of the spiked sample as template in the same manner

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as described above.

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Statistical analyses

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For the relationships between the counts of eDNA detection and eDNA concentrations, we fit

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generalized linear models (GLMs) with binomial error distributions to eDNA counts from ponds

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and LOD samples for each PCR method [eDNA count from eight replicates] = log10 [eDNA

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copies]. To compare the differences in eDNA counts among PCR methods, we fit a GLM with

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binomial error distribution, including an eDNA × PCR method interaction, [eDNA count from

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eight replicates] = log10 [eDNA copies]*[PCR types].

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To compare eDNA presence in ponds among PCR types, we also fit GLMs with binomial

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error distributions in the same manner as above [eDNA presence of ponds (0 or 1)] = log10


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[eDNA copies] and [eDNA presence of ponds (0 or 1)] = log10 [eDNA copies]*[PCR types]. All

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statistical analyses were performed at a significance level of α = 0.05 using R ver. 3.1.0.39

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Results

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Detection of pond samples

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There were significant positive relationships between DNA concentrations, which were

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measured by real-time PCR with GEMM in Takahara et al.15, and the detection rate for all three

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PCR methods (GLM, p < 0.001, Fig. 2). In pond samples, which had more than 150 copies L–1,

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bluegill DNA was detected in all replicates by all PCR methods. ddPCR and real-time PCR with

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EMM detected bluegill DNA in 3 and 1 pond sample in which the target DNA was not detected

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by real-time PCR with GEMM, respectively (Fig. 3). In contrast, real-time PCR with EMM did

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not detect the target DNA in one pond sample in which target DNA was detected by real-time

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PCR with GEMM. The parameters of GLMs were significantly different among PCR types

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(GLM, |z| > 3.094, p < 0.001 for all coefficients; DNA concentrations, PCR methods, and DNA

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concentration × PCR method, Fig. 2), indicating that the slopes of GLMs differed significantly

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among PCR methods, but the trends were associated with DNA concentration. ddPCR had

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higher detection rates than real-time PCR with GEMM or EMM at low DNA concentrations.

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However, the ddPCR detection rate was lower than that of real-time PCR at moderate DNA

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concentrations (i.e., 3-5 DNA copies).

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There were significant positive relationships between DNA concentration, as measured by

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ddPCR, and detection probability (GLM, p < 0.001, Fig. 3). GLM parameters were not


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significantly different among the PCR methods (GLM, |z| < 1.815, p = 0.064 for all coefficients:

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DNA concentration, PCR method, and DNA concentration × PCR method, Fig. 3).

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LOD analysis

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1) DNA dilution experiment; The LOD trends (i.e., detection rates as a function of number of

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DNA copies) did not differ significantly among PCR methods (GLM, z = 0.557, p = 0.578, Fig.

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4). However, when using 2 and 3 DNA copies, real-time PCR with GEMM and EMM had

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slightly higher detection rates than ddPCR. In all PCR types, the lower limit of detection was at 1

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copy per reaction, and the detection rates tended to increase with increasing DNA concentration.

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2) DNA spiking experiment; The LOD trends differed significantly among PCR methods

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(GLM, z = 2.156, p = 0.0301, Fig. 5), indicating that ddPCR had higher detection rate for target

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DNA than real-time PCR in field samples containing inhibitors. At target DNA concentrations of

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5 and 10 copies per reaction, ddPCR had higher detection rates than real-time PCR. Only ddPCR

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detected target DNA in all 8 replicates at the concentration of 10 DNA copies/reaction (Fig. S1).

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In all PCR types, the lower limit of detection was 1 copy per reaction, and the detection rates

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tended to increase along with increased DNA concentration.

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Discussion

239 240

We found that eDNA detection rates using ddPCR were higher than those obtained using real-

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time PCR with either GEMM or EMM, particularly in field samples with low DNA

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concentrations. Real-time PCR with EMM had higher eDNA detection rates than real-time PCR


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with GEMM. Thus, in general, detection rates for ddPCR were greater than those for real-time

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PCR with EMM, which in turn were greater than those for real-time PCR with GEMM.

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However, there was little difference in LOD results among the PCR methods.

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One of the main reasons for higher eDNA detectability using ddPCR and real-time PCR with

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EMM is likely to be that these PCR methods decrease PCR inhibition by compounds in the

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natural environment. In ddPCR, each droplet contains 0 or 1 to few copies target DNA. This

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distribution would be also true for inhibitors, which can reduce concentrations of PCR inhibitors

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in ddPCR than present in real-time PCR.36 From our DNA spiking LOD test, ddPCR had higher

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detection rate than real-time PCR and supported our explanation for PCR inhibitor effects on

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detectability. Furthermore, ddPCR uses end-point PCR amplification in each nano-liter droplet to

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independently detect amplified DNA amplification. Such methodological advantages of ddPCR

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increase the detectability of DNA in field samples in the face sample-dependent limitations. In

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real-time PCR, the tolerance of EMM to PCR inhibitors was greater than that of GEMM. Such

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tolerance to PCR inhibitors should allow detection of DNA in samples with high concentrations

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of PCR inhibitors. Thus, real-time PCR with EMM had higher detection rates than real-time PCR

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with GEMM.

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In an assessment of the effect of sample volume on PCR with GEMM, Takahara et al.11

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suggested that 2 µL samples showed a smaller PCR inhibition effect on eDNA detection than 5

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µL samples. However, using ddPCR or EMM may allow the PCR sample volume to be increased

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to 5 µL or more (e.g., a previous study has used up to 13 µL eDNA sample in 20 µL PCR

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reaction. 40). We were unable to test the effects of sample volume due to limited available sample

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solution (100 µL), but future studies should consider estimating eDNA detection rates using

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ddPCR and real-time PCR with EMM with larger PCR sample volumes.


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The ddPCR detection rate was higher than that of the other PCR methods at low DNA

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concentrations, but was slightly lower than that of real-time PCR at moderately low DNA

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concentrations (e.g., 2-5 copies per PCR reaction). The relationship between detection rates and

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DNA concentration was more linear for ddPCR than for real-time PCR. One possible

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explanation would be that the detection mechanism of ddPCR is more resistant to external

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factors, such as the effects of inhibitory substances, or low eDNA concentrations. Unlike in the

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detection of end-point amplification in each droplet, as in ddPCR, real-time PCR can be easily

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adversely affected by low amplification efficiency because it measures the fluorescence intensity

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of amplicons in 20-µL PCR reactions. However, to further our understanding of the underlying

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mechanisms, further studies would be necessary.

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There has been increasing attentions to false positive and negative eDNA detection rates.41

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Recently, a model for estimating false positive and negative rates of detection, while accounting

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for eDNA sampling effort, has been proposed.42 ddPCR and the use of EMM in real-time PCR

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assays have the potential to decrease false negative eDNA detection rates without increasing

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sampling effort. ddPCR and EMM may contribute to increased certainty in not only eDNA

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studies, but also in the development of species distribution and occupancy models, which are

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widely applied to population management.42 In this study, we were unable to test false positive

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DNA detection rates, although we did not find any DNA copies in the negative controls. More

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intensive analysis using negative samples with/without spiking with target DNA were needed to

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test false-positive detection rates for ddPCR and real-time PCR.

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eDNA techniques have been applied to estimate species’ biomass and abundance.10, 20, 34, 35 To

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accurately estimate eDNA concentrations in field samples, PCR platforms amenable to very low

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template DNA concentrations are required. ddPCR was more accurate in quantifying fish eDNA


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at low concentrations than real-time PCR, 35 and similar results were reported in a comparison of

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ddPCR and real-time PCR using HIV DNA.30 It seems ddPCR can quantify even a few DNA

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copies with high precision. Thus, ddPCR is a useful PCR platform due to its ability to for

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estimate eDNA concentrations with higher precision than real-time PCR and detect eDNA

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presence at low concentrations.

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Taken together, our results suggest ddPCR is a better platform than real-time PCR for

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detecting eDNA and quantifying its abundance in field samples. However, the disadvantages of

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the three PCR methods should also be considered. 1) Cost and time: conducting ddPCR assays is

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more expensive and time-consuming than conducting real-time PCR,35 and ddPCR facilities are

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much more expensive than those for real-time PCR. The cost for real-time PCR is similar for

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either GEMM or EMM because the prices of the two reagents are almost the same. 2) Carry-over

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contamination risk: to minimize carry-over contamination of PCR products, GEMM contains a

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Uracil-DNA Glycosylase, while ddPCR supermix and EMM do not. Thus, using GEMM in real-

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time PCR can mitigate carry-over risks compared to ddPCR and real-time PCR with EMM. In

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this study, we did not find any carry-over effects in any PCR platform (i.e. did not detect DNA in

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any negative controls). However, future studies should test for carry-over effects in ddPCR and

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real-time PCR with EMM assays. 3) Amplification and detection mechanisms: for ddPCR, an

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appropriate threshold setting is essential for detection of eDNA. With a default fluorescence

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threshold setting, such as >2000, we could not detect droplets containing DNA in some cases

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(e.g., at lower DNA concentrations) and in such cases the detection rate of ddPCR was less than

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that of real-time PCR. In this study, we manually set the threshold used in LOD analysis

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according to outputs obtained from blank samples and those with only a few DNA copies. Some

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of the samples in this study only had a few positive droplets in ddPCR. Such low number of


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positive droplets can potentially increase false-positive detection rates. However, we did not find

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any positive droplets from all negative controls (32 negative controls in total). Thus, the samples

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with positive droplets using the threshold of 1100 RFU would have contained the target DNA.

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Although further experiments are required to closely examine the optimum fluorescence

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threshold to detect eDNA in ddPCR, the manual adjustment using negative control samples and

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those with a few DNA copies would be one of the options to determine the threshold. In

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addition, after considering preliminary tests and the maximum Ct value of real-time PCR (40.8),

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we used 45 PCR cycles for ddPCR, which is less than the number used for real-time PCR (55

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cycles). Despite this, the detection rates of ddPCR in field samples were higher than those of

321

real-time PCR. Because ddPCR measures the end-point of PCR amplification, we could not

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check the amplification process (i.e., amplification plots and resulting amplicons). This is a

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disadvantage of ddPCR; we could not identify non-target DNA amplification or other problems

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occurring during PCR cycles. The number of PCR cycles for ddPCR should be carefully

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determined with reference to the Ct values of real-time PCR or other information.

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In conclusion, ddPCR is superior to real-time PCR for detecting eDNA in water from natural

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habitats, such as ponds, especially at low DNA concentrations. In this study, we used aquatic

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samples; DNA samples from soil and sediment generally contain more PCR inhibitory

329

substances.19 Thus, in other ecosystems, ddPCR would likely also have higher eDNA detection

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rates than real-time PCR, which could help to better assess species distributions, but this needs

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confirmation.

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AUTHOR INFORMATION

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Corresponding Author


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Institute for Sustainable Sciences and Development, Hiroshima University,

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1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8530 Japan

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E-mail: [email protected]

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Tel: +81-82-424-5732

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Fax: +81-82-424-5732

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Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval

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to the final version of the manuscript. ‡These authors contributed equally.

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Funding Sources

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Environment Research and Technology Development Fund (4RF-1302)

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Industry-University Collaboration Grant for Young Scientists in Hiroshima University

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JST-CREST

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ACKNOWLEDGMENT

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We sincerely thank Dr. Keiichi Fukaya, The Institute of Statistical Mathematics, who gave

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advice on our experimental design and statistical analyses. We sincerely thank the Faculty of

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Science and the Natural Science Center for Basic Research and Development, Hiroshima

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University, for providing PCR facilities. This study was supported by the Environment Research


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and Technology Development Fund (4RF-1302) of the Ministry of the Environment, Japan, JST-

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CREST, and in part by the Industry-University Collaboration Grant for Young Scientists in

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Hiroshima University to Teruhiko Takahara.

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Supporting Information Available

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Appendix S1 Testing for PCR cycle number for ddPCR

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This information is available free of charge via the Internet at http://pubs.acs.org/ .

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FIGURES

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Figure 1. Fluorescence amplitude (FAM) of droplets in samples of pond water (3/8 detected,

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right plot) and negative control (DNA-free water, left plot), output from QuantaSoft software for

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ddPCR. Pink line indicates the threshold to detect DNA at 1100 RFU. Blue and black dots mean

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positive and negative droplets. Yellow dotted line separates each replicate.

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Figure 2. Detection rate of bluegill eDNA in pond water for three PCR methods; real-time PCR

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with GEMM, EMM and ddPCR. The blue lines indicate the binomial GLM results.

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Figure 3. Bluegill eDNA presence in the ponds for three PCR methods; real-time PCR with

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GEMM, EMM and ddPCR. The blue lines indicate the binomial GLM results.

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Figure 4. Detection rates in limited of detection (LOD) analyses using DNA dilution experiment

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for three PCR methods; real-time PCR with GEMM, EMM and ddPCR. The blue lines indicate

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the binomial GLM results.


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Figure 5. Detection rates in limited of detection (LOD) analyses using DNA spiking experiment

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for three PCR methods; real-time PCR with GEMM, EMM and ddPCR. The blue lines indicate

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the binomial GLM results.

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