Filtration extraction method using microfluidic channel for
measuring environmental DNA
[Running title: Microfluidic filtration method for eDNA]
Takashi Fukuzawa1,2,*, Yuichi
Kameda1,3, Hisao Nagata1, Naofumi
Nishizawa1,
Hideyuki Doi4,*
1 GO!FOTON INC., Tsukuba, Japan
2 Biryu Planning, Tokyo, Japan
3 Department of Anthropology, National Museum of Nature and Science,
Tsukuba, Japan
4 Graduate School of Information Science, University of Hyogo, Kobe,
Japan
*Co-corresponding authors:
Takashi Fukuzawa, Email:takashi.fukuzawa@gofoton.com
Hideyuki Doi, Email:hideyuki.doi@icloud.com
Abstract
The environmental DNA (eDNA) method, which is widely applied for
biomonitoring, is limited to laboratory analysis and processing. In this
study, we developed a filtration/extraction component using a
microfluidic channel, Biryu-Chip (BC), and a filtration/extraction
method, BC method, to minimize the volume of the sample necessary for
DNA extraction and subsequent PCR amplification. We tested the
performance of the BC method and compared it with the Sterivex
filtration/extraction method using aquarium and river water samples. We
observed that using the BC method, the same concentration of the
extracted DNA was obtained with 1/20–1/40 of the filtration volume of
the Sterivex method, suggesting that the BC method can be widely used
for eDNA measurement. In addition, we could perform on-site measurements
of eDNA within 30 min using a mobile PCR device. Using the BC method,
filtration and extraction could be performed easily and quickly. The PCR
results obtained by the BC method were similar to those obtained by the
Sterivex method. The BC method required fewer steps and therefore, the
risk of DNA contamination could be reduced. When combined with a mobile
PCR, the BC method can be applied to easily detect eDNA within 30 min
from a few 10 mL of the water sample, even on-site.
Keywords: eDNA, extraction, filtering, microfluidic
Introduction
Environmental DNA (eDNA) in aquatic environments has been used to detect
the distribution of the species
(Doi et al., 2017a; Takahara,
Minamoto, Yamanaka, Doi, and Kawabata, 2012; Katano, Harada, Doi, Souma,
and Minamoto, 2017). eDNA has been detected in water from various
ecosystems, including streams, lakes, ponds, reservoirs, canals, lakes,
and oceans (Doi et al., 2017;
Takahara, Minamoto, Yamanaka, Doi, and Kawabata, 2012; Katano, Harada,
Doi, Souma, and Minamoto, 2017; Fornillos et al., 2019). eDNA
measurements have been mainly performed using quantitative real-time PCR
(qPCR) (Doi et al., 2017a; Takahara, et al., 2012; Katano et al. 2017;
Fornillos et al., 2019). However, it is limited to laboratory analysis,
which usually takes several hours. These time delays often limit the
range of uses for on-site eDNA detection (Thomas et al., 2020; Nguyen et
al., 2018). Field-portable DNA extraction and PCR platforms offer a way
for species detection by eDNA analysis on-site (Thomas et al., 2020;
Nguyen et al., 2018; Thomas, Howard, Nguyen, Seimon, and Goldberg,
2018). However, this approach takes a similar time as that of the
laboratory measurements. Recently, Doi et al. (2021) reported a simpler
extraction method in the field using PicoGene PCR1100 (mobile qPCR;
Nippon Sheet Glass, Sagamihara, Japan), which allowed eDNA detection in
a relatively short time (Doi et al., 2021). This method required
approximately 30 min to complete after sample water collection for DNA
measurement by qPCR. However, time and facilities are limited for sample
analysis in the field, and there is a need for a simpler and more
efficient pretreatment method, including filtration. In particular, the
simplicity of species-specific eDNA measurement should be one of the key
features to allow for the large-scale implementation of eDNA technology.
In eDNA measurements, the sensitivity of DNA detection is important.
Therefore, it is essential not to compromise on the concentration and
yield of the extracted DNA from the water samples. Although various
extraction methods using DNA isolation kits such as the DNeasy Blood and
Tissue Kit (Qiagen, Hilden, Germany) (Tsuji et al., 2019), PowerWater
(Hinlo, Gleeson, Lintermans, and Furlan, 2017; Coster, Dillon, Moore,
and Merovich, 2021), and PowerSoil (Sakata et al., 2020; Eichmiller,
Miller, and Sorensen, 2016; Díaz et al., 2020) have been used, it is
necessary to simplify the process from water sampling to eDNA extraction
for field measurements and general users.
They include disc-shaped filters (e.g., GF/F glass filters) (Tsuji et
al., 2019), nucleopore filters (e.g., 0.2-µm pored filter) (Takahara et
al. 2012; Sassoubre, Yamahara, Gardner, Block, and Boehm, 2016), and
cartridge filters (e.g., Sterivex) (Miya et al., 2016). However, if less
volume of the DNA extraction solvent is used, it is difficult to contact
the entire area of the filter uniformly. As a result, DNA would
partially remain on the filter. Consequently, the DNA yield would
decrease. In contrast, when a microfluidic channel is used for
filtration, the entire surface of the filter on the channel comes in
contact with the DNA extraction solvent. Accordingly, DNA can be
extracted efficiently and a high concentration of the extracted DNA can
be obtained.
In this study, we developed a filtration/extraction component using a
microfluidic channel to minimize the volume of the sample necessary for
DNA extraction, simultaneously yielding a high concentration of eDNA for
subsequent PCR amplification. We demonstrated that eDNA can be measured
easily with low contamination risk using this component (Muha, Robinson,
Garcia de Leaniz, and Consuegra, 2019). We also tested the performance
of the developed microfluidic channel using samples from aquarium and
river water and compared the results with conventional methods using
Sterivex filters, DNeasy Blood and Tissue Kit, and benchtop qPCR for
various fish species.
Materials and Methods
Material design for microfluidic channel chip: Biryu-Chip
We developed a filtration/extraction chip with a microfluidic channel,
called the Biryu-Chip (Biryu: micro-channel in Japanese) and the
filtration/extraction method is termed as the Biryu-Chip (BC) method. A
schematic diagram of the BC is shown in Fig. 1a, and a photograph is
shown in Fig. 1b. The base material was made of cycloolefin polymer and
molded by injection molding. The filter was made of polyvinylidene
difluoride, the same material as the Sterivex filter. The upper channel
of the filter is 1 mm wide (i.e., the effective width of the filter),
0.5 mm high, and 40 mm long, with a cross-sectional area of 0.5
mm2. During filtration, the sample was passed from the
inlet to the outlet using a syringe and the eDNA was trapped on the
filter (Fig. 1c). Finally, the DNA was extracted into the buffer
(Fig.1d).
Validation experiments
Four experiments were performed to validate the BC method. Experiment 1:
determining the volume of DNA extraction solvent for the BC method by a
benchtop qPCR using water samples spiked with rainbow trout
(Oncorhynchus mykiss ) aquarium water; 2: comparing the BC method
with the Sterivex method by a benchtop qPCR analysis (hereafter referred
to as Sterivex-qPCR) using water samples spiked with rainbow trout
aquarium water; 3: field validation of the BC method performed using the
river water samples and compared with the results using the Sterivex
method by detecting sweetfish (Plecoglossus altivelis ) DNA on a
benchtop qPCR; and 4: field validation of the BC method using a mobile
PCR (hereafter referred to as BC-mobile PCR) was done from another
sample of river water which was a habitat to the common carp
(Cyprinus carpio ). The outline of the extraction steps of the
Sterivex and BC methods is shown in Fig. 2.
Experiment 1
We collected 4 mL of rainbow trout aquarium water and made up the final
volume to 200 mL with pure water, mixed well, and then filtered 10 mL of
diluted sample each through eight BCs. Extraction of the DNA was
performed according to the BC method. Different volumes of the
extraction solvent, as explained later, were used for DNA extraction in
increasing volume, that is 6, 8, 10, 12, 14, 16, 18, and 20 µL
representing eight levels. We measured the concentration of the
extracted DNA using a benchtop qPCR, as described below.
Experiment 2
We collected 2 mL of rainbow trout aquarium water and made up the volume
to 250 mL with pure water and mixed well. Then, 10 mL of the diluted
sample was filtered by the BC filter and 200 mL by the Sterivex filter.
DNA extraction from the BC filter was done using 20 µL of the extraction
solvent. DNA from the Sterivex filter was extracted using a DNeasy Blood
and Tissue Kit. We measured the concentration of DNA extracted by both
methods using a benchtop qPCR.
Experiment 3
We collected 250 mL of surface river water from the middle basin of the
Sagami River (35.575099°N, 139.308802 °E), a known sweetfish habitat.
Two water samples (10 mL each) were filtered by two BC filters and 200
mL was filtered by the Sterivex filter. DNA from the BC filter was then
extracted with 10 and 20 µL of the extraction solvent. DNA from the
Sterivex filter was extracted using a DNeasy Blood and Tissue Kit. We
measured the concentration of DNA extracted by the BC and Sterivex
methods using a benchtop qPCR.
Experiment 4
In this experiment, we used eDNA of the common carp from the Sakai
River. We collected water samples at several points (from bridge 1:
35.600385°N, 139.354699°E to bridge 6: 35.604273°N, 139.335399°E) in the
Sakai River (Fig. 3) using a centrifuge tube attached to a fishing line.
The sample water (10 mL) was filtered through the BC chip, resulting in
an extraction volume of 20 µL.
Filtration and DNA extraction by the BC method
We performed sample filtration and DNA extraction by the BC method using
the setup shown in Fig. 4. The procedure followed is given below:
1) Water was collected using a disposable container, such as a 50 mL
centrifuge tube. A syringe (e.g., 10 mL volume) was used to aspirate a
predetermined volume of water and then the water sample was injected
into the BC inlet and filtered.
2) The syringe was removed after sample filtration. The plunger was
pulled to draw air and then the syringe was inserted into the BC inlet.
Then, the plunger was pushed to drain the excess water from the chip.
3) The seal attached to the extraction port of the BC filter was
removed. The extraction solvent was then injected into the filter (10
and/or 20 µL for Experiment 2–4, 6–20 µL for Experiment 1) and mixed
with a pipette to allow the movement of the extraction solvent
throughout the upper channel of the filter for 2 min. Solution A of the
Kaneka Easy DNA Extraction Kit (Version 2) (Kaneka, Tokyo, Japan) was
used as the extraction solvent.
4) The extraction solvent was drawn from the extraction port using a
pipette and then transferred to a microcentrifuge tube to obtain the
extracted DNA sample.
DNA extraction by the conventional method
To evaluate the performance of the BC method in Experiments 2 and 3,
we compared its results with that of the conventional method, which is a
combination of the filtration method using the Sterivex filter
(Takahara, Minamoto, Yamanaka, Doi, and Kawabata, 2012; Sassoubre,
Yamahara, Gardner, Block, and Boehm, 2016) and the extraction method
using the DNeasy Blood and Tissue Kit (Tsuji et al., 2019). Filtration
using the Sterivex filter is relatively easy to perform on-site without
the requirement of sophisticated infrastructure and the DNA extraction
method using the DNeasy Blood and Tissue Kit is a widely used method in
academic research (Minamoto et al., 2021). Filtration and DNA extraction
from the water samples collected in the experiments above were performed
according to the manufacturer’s instructions (Minamoto et al., 2021).
PCR kit used for benchtop qPCR and mobile PCR
It is known that the efficiency of PCR amplification decreases in the
presence of substances such as humic acid, which is a PCR inhibitor
(Uchii et al., 2019). Since the purification of the water sample was not
involved in the BC method, the effect of PCR inhibitors may be
significant. Therefore, in this study, we used KAPA3G Plant PCR Kit
(Sigma-Aldrich, Missouri, USA) which is relatively resistant to PCR
inhibitors.
Benchtop qPCR
Quantification of eDNA from all the samples except those in Experiment 4
was done using the StepOnePlus Real-Time PCR System (Thermo Fisher
Scientific, Massachusetts, USA). For the laboratory qPCR analyses, we
used the same set of primer-probe as in the on-site measurements.
Similarly, the PCR template mix used was as described in the previous
studies (Doi et al., 2017a, Uchii et al., 2019). Each TaqMan reaction
contained 900 nM of each primer (forward and reverse), 125 nM
TaqMan-Probe, 0.03 U/µL qPCR master mix (KAPA3G Plant PCR Kit), and 1.5
μL of the eDNA solution. The final volume for a single PCR assay was made
up to 15 μL by adding distilled water (DW). The volume of the eDNA
solution added was 10% of the volume of the PCR mixture. The qPCR
conditions were as follows: 95 °C for 20 s, followed by 55 cycles of 95
°C for 4 s and 60 °C for 20 s. Three replicates were performed for each
sample and no-template control (NTC). Standard curves of qPCR
measurements had R2 = 0.995–0.998 and PCR efficiency
= 95.4%–105.2%.
Mobile PCR
Before sampling, we prepared a PCR pre-mix with preliminary mixing of
the master mix and primer probe. This pre-mix was brought on-site for
PCR analysis. Each TaqMan reaction contained 900 nM of each primer
(forward and reverse), 400 nM of TaqMan-Probe, 0.1 U/µL qPCR master mix
(KAPA3G Plant PCR Kit). The final volume for PCR pre-mix was made up to
14.4 μL by adding DW. Then, 1.6 μL of the eDNA solution was added to the
tube containing the pre-mix. The volume of the eDNA solution added was
10% of the volume of the PCR mixture. This was followed by the
injection of this solution into the flow path of the PicoGene PCR1100.
The PCR pre-mix was stored in a cooler at 5 °C until PCR was performed.
The mobile PCR conditions were as follows: 95 °C for 15 s, followed by
50 cycles of 95 °C for 3.5 s, and 60 °C for 10 s. Additionally, to check
the cross-contamination in the PCR machine on-site, we performed an NTC
using DW after completion of all the PCR measurements for the day (PCR
control) on site. The primer-probe sets are listed in Table 1.
Statistical analysis
All statistical analyses were performed using the R package (ver. 4.1.1)
(R Core Team, 2021) and the ggplot2 package. The significance level was
set to α = 0.05. We performed a simple linear model to predict the DNA
extraction volume of the BC method and the DNA concentration using the
”lm” function. For categorical data of Experiments 2 and 3, we performed
one-way ANOVA using the ”aov” function. When the ANOVA was significant,
we performed the post-hoc test by Tukey’s multiple comparisons using the
”TukeyHSD” function for comparing each difference.
Results
Experiment 1: Determination of the volume of the extraction solvent
Fig. 5 shows DNA extraction by the BC method and qPCR results (DNA copy
number) using different volumes of the extraction solvent. From the
linear model results, we observed a significant relationship between the
volume of the extraction solvent and DNA copy number
(R2 = 0.9167, P for R2 and slope
< 0.0001).
Experiment 2: Comparison using the samples spiked with aquarium water
Fig. 6 shows the qPCR results of the amplification of DNA from the
rainbow trout in the samples extracted by the Sterivex and BC methods.
We observed significant differences in ANOVA (F= 11.65, P <
0.0001) and Tukey’s comparison (P < 0.05) for the pair of
Sterivex 1 and BC 1 (Fig. 6). The average copy number of the four
extracted samples were 385 ± 141 copies µL-1 for the
Sterivex method and 599 ± 154 copies µL-1 for the BC
method. The coefficient of variation (CV: standard deviation/mean value)
of the Sterivex and BC method were 0.33 and 0.26, respectively,
indicating that the BC method did not show a large variability. These
results suggest that the eDNA concentration extracted using the BC
method from various samples was similar despite a lower filtration
volume (5%). In the negative control, no rainbow trout DNA was detected
in the extracted sample of non-spiked water in each method.
Experiment 3: Comparison with filed samples
The water of the Sagami River was used in this experiment. We observed
differences in the concentration of DNA from the sweetfish as determined
by the Sterivex and the BC method using 10 and 20 µL of extraction
solvents (Fig. 7). We also observed that ANOVA for the three categories
was significant (F = 55.66, P < 0.0001). The BC method using
10 µL of the extraction solvent had significantly highest DNA
concentration as determined by the Tukey’s test (P < 0.0001).
The mean value of the copy number from extracted samples for each
category was 34 ±6.7 copies µL-1 for the Sterivex
method, 38 ± 12.3 copies µL-1 for the BC method with
20 µL of the extraction solvent, and 82 ± 16.5 copies
µL-1 for the BC method with 10 µL of the extraction
solvent. The CV values of the Sterivex method, BC method with 20 µL and
10 µL of the extraction solvent were 0.18, 0.30, and 0.19, respectively.
Experiment 4: eDNA measurement in the field
Fig. 8 shows the Ct values of carp eDNA obtained by the on-site
BC-mobile PCR methods. We detected high concentrations of the common
carp DNA at all survey sites. From these results, it could be inferred
that the common carp was living in river water between the bridges 5 and
6.
Discussion
In this study, we developed a new filtration/extraction method called
the BC method for eDNA measurements from various water ecosystems. We
observed that using this method, the concentration of DNA from the
extracted samples was almost similar to the concentration of DNA
extracted using the conventional methods even with 1/20–1/40 of the
filtration volume that is generally used in the Sterivex method.
Therefore, the BC method can be used to obtain a highly concentrated DNA
sample even with low filtration volumes. Although the efficiency of the
BC method may change according to the target eDNA, it is not inferior to
the conventional methods in terms of the DNA yield, suggesting that it
can be widely used for eDNA measurement.
As the filtering volume of BC method is small while measuring eDNA at
lower concentrations, the volume of eDNA to be filtered is also small.
As a result, the variation may be larger than when using a large volume
of sample water which may affect the detection limit. This problem with
the detection limit could be solved by further increasing the
concentration ratio by optimizing the filtration and extraction
conditions of the chip. In addition, even if the detection limit is
reduced, it can be expected that the BC method has industrial
applications where it can be used by balancing the merits of
simplification.
In the BC method, the volume required for filtration can be greatly
reduced, and the method makes water sampling simple. For example, in the
design of the BC method, it requires less than 25 mL filtration volume
compared to 0.5 to a few liters of water for the previous methods (Tsuji
et al., 2019). For measurements of eDNA from the habitats where it is
difficult to collect water, such as small wetlands (Doi et al., 2017b),
the use of the BC method would be preferable. The process after
filtration in the BC method is much simpler than the Sterivex method and
does not require a high level of skill. The working time was reduced
from 90 min to 3 min. In addition, special equipment such as centrifuges
and rotators are not required, which makes it easier to introduce
measurement equipment in the process. As a result of the simplification
of filtration and extraction, a set of components and instruments from
water sampling to PCR can be stored in an attached case (Fig. 4d), and
eDNA detection can be easily performed on-site in approximately 30 min,
like our previous method (Doi et al. 2021).
The situations in which these advantages are of high importance include
on-site eDNA measurement and in study areas where it is difficult to
collect water such as wetlands and valleys. The BC-mobile PCR method has
the following benefits: measurements at multiple points in a day and the
sampling site which is difficult to access.
The filtration and extraction steps of the eDNA measurement has the
risk of contamination. Moreover, the risk is also higher if there are
several reusable components, such as filtering and extraction equipment,
including a filter funnel and centrifuge machine. However, the BC method
uses disposable materials, including the chip, thus reducing the
contamination risk in the eDNA measurements. In addition, since
filtration and extraction can be performed consistently in the closed
space of the BC, there are fewer opportunities for the samples and
reagents to be exposed to the surrounding air. This further reduces the
risk of contamination compared to other filtration and extraction
methods. Furthermore, the principal of the BC method can be used for
detecting bacteria or viruses by changing the design of the BC and
filter. Thus, the BC method can be applied to broad scientific fields of
research, such as the human health and food sciences.
In conclusion, we developed the Biryu-Chip, a filtration/extraction
component, and a method for measuring eDNA using a microfluidic channel.
Using the BC method, filtration and extraction can be performed easily
and quickly, and it was demonstrated that similar results can be
obtained with approximately 1/20–1/40 of the filtration volume compared
to the Sterivex method. Since the BC method requires fewer steps, the
risk of contamination, which is a problem in eDNA measurements, would be
reduced. When combined with a mobile PCR device, this method could
detect eDNA within approximately 30 min even on-site. Since a variety of
water samples with different turbidity and species are handled in eDNA
measurements, it is necessary to validate the usefulness of this method
at multiple sites, including samples with more foreign substances, in
the future. However, in some cases, it may be necessary to consider the
selection of PCR reagents that are more resistant to PCR inhibitors.
Data availability
All data (Supplementary Table S1) are available in Zenodo (doi:
10.5281/zenodo.5709221).
Acknowledgments
This study was supported by the Environment Research and Technology
Development Fund (JPMEERF20204004).
Conflict of interest
The commercial affiliations of authors [TF, NN, HN, and YK] do not
alter our adherence to the journal policies on sharing data and
materials. TF, NN, HN, and YK were employed by the manufacturer of the
equipment described. However, none of the authors will directly benefit
from the publication of this paper.
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