Pakistan J. Zool., pp 1-10, 2021.

DOI: https://dx.doi.org/10.17582/journal.pjz/20190118100108

An Optional Low-Cost Method of Extracting
Environmental DNA of Macro-Organisms from
Filter Membranes in Large Scale eDNA Surveys
Zhi Chen1, Toshifumi Minamoto2, Longshan Lin3 and Tianxiang Gao4,*
College of Fisheries and Life Science, Hainan Tropical Ocean University, Sanya
572022, China
2
Graduate School of Human Development and Environment, Kobe University, Kobe
657-8501, Japan
3
Marine Life and Ecology Laboratory, Third Institute of Oceanography, State
Oceanic Administration, Xiamen 361005, China
4
Fisheries College, Zhejiang Ocean University, Zhoushan 316022, China
1

Environmental DNA (eDNA) analysis is a powerful tool within ecology for the distribution or abundance
study of aquatic species. DNA extracting was an indispensable process to employ this method. In eDNA
extracting of macro-organisms, DNeasy Blood and Tissue Kit was commonly used, but its price wasabout 80 RMB/sample, which was too expensive. Cheaper eDNA extraction kit was a potential aspect
for improvement to expand further application of eDNA analysis. In order to examine some cheaper kits’effectiveness of extracting eDNA of macro-organisms and select an optional low-cost kit, a test among
three kits was held. 12S rRNA bands of marine fishes on the agarose gel preliminarily revealed that all
three kits could successfully extract eDNA from field water samples. Digital PCR results further revealed
that the average eDNA concentration of Sepiella japonica extracted by three kits were (4.62 ± 0.72) ×
105, (3.83 ± 0.56) × 105, and (2.35 ± 0.27) × 105 copies/300 ml, respectively. The eDNA concentrations
by AxyPrep DNA Gel Extraction Kit were about 83% of that extracted by DNeasy Blood and Tissue Kit.
However, extracting cost of an eDNA sample using AxyPrep DNA Gel Extraction Kit was about 3.75% ofthat using DNeasy Blood and Tissue Kit. Up to now, many eDNA surveys have collected eDNA samples
with large scale, which sometimes required hundreds of filter membranes and a mass of extraction kits.
Combined the price and effectiveness, using AxyPrep DNA Gel Extraction Kit as an substitute for DNeasy
Blood and Tissue Kit could remarkably cut the eDNA extracting cost. AxyPrep DNA Gel Extraction Kit
might be an optional low-cost method of extracting environmental DNA of macro-organisms from filter
membranes in large scale eDNA surveys.

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INTRODUCTION

E

nvironmental DNA (eDNA) represented all types of
DNA present in the environment, DNA from organisms
themselves and extracellular DNA (Ficetola et al., 2008;
Wilcox et al., 2013). eDNA approaches for mapping
the distribution of organisms were first used for microorganisms (Lydolph et al., 2005; Gulraiz et al., 2017).
Recently, they have been applied to macro-organisms(Ficetola et al., 2008; Jerde et al., 2011; Minamoto et al.,2012). It was estimated that macro-organisms released
their DNA via egestion, excretion, secretion, exfoliation,
reproduction and decomposition (Pilliod et al., 2013;
Minamoto et al., 2017), and that the main form of the eDNA
would be derived from organelles (Santas et al., 2013;*
Corresponding author: gaotianxiang0611@163.com
0030-9923/2021/0001-0001 $ 9.00/0
Copyright 2021 Zoological Society of Pakistan

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ABSTRACT

Article Information
Received 18 January 2019
Revised 02 March 2019
Accepted 13 March 2019
Available online 25 June 2020
Authors’ Contribution
ZC and TG presented concept andanalyzed the data. ZC, TM, LL
and TG wrote the manuscript. TG
supervised the study and arranged
financial support.
Key words
Environmental DNA, DNA extraction
kits, Sepiella japonica, Effectiveness,
Cost.

Turner et al., 2014). The presence or absence of the eDNA
for a specific organism generally indicated the presenceor absence of that organism (Turner et al., 2014; Biggs etal., 2015), and the eDNA concentration was considered toreflect the biomass or abundance of the target organisms(Takahara et al., 2012; Yamamoto et al., 2016; Doi et al.,2017). To date, eDNA surveys of macro-organisms havebeen conducted in different aquatic systems (Thomsen
and Willerslev, 2015; Doi et al., 2015b), such as wetlands(Piaggio et al., 2014), tanks (Kelly et al., 2014; Minamoto
et al., 2017), streams (Goldberg et al., 2011; Jane et al.,2014; Sigsgaard et al., 2015), rivers (Goldberg et al., 2013;
Deiner and Altermatt 2014), ponds (Dejean et al., 2012;
Treguier et al., 2014; Takahara et al., 2015), lakes (Eganet al., 2013; Jerde et al., 2013; Eichmiller et al., 2014) andoceans (Foote et al., 2012; Pilliod et al., 2014; Valentini
et al., 2016). Although this new method was reported tooutperform the traditional survey methods including directcatch by angling and netting, direct observation by diving,Z. Chen et al.

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and indirect observation by filming and echo sounding(Olson et al., 2013; Thomsen and Willerslev, 2015;
Minamoto et al., 2017), there were still many problems
that needed improvement.
For example, to get more reliable data about macroorganisms, researchers sometimes collected eDNA
samples in triplicate at each site (Margaret et al., 2015;
Matthew et al., 2015; Merkes et al., 2015), which would
no doubt increase the cost of eDNA collecting. Besides,
DNeasy Blood and Tissue Kit (Qiagen) were universallyused to extract eDNA from filter membranes (Takahara
et al., 2013; Thomsen and Willerslev, 2015; Ikeda et al.,2016; Minamoto et al., 2017). This kit provided fast andeasy silica-based DNA purification in convenient spincolumn and 96-well-plate formats. Most tissue and blood
samples can be normally extracted according to operating
instruction. However, double or more dosage of buffer AL
was used because of the obvious water-absorbing quality offilter membranes (Takahara et al., 2012; Doi et al., 2015a,b; Fukumoto et al., 2015; Thomsen and Willerslev, 2015).
The price of DNeasy Blood and Tissue Kit (50 times) was
usually 2000 RMB, double dosage and triplicate meant
approximate 80 RMB/sample and only 8 or 9 samplingsites that such a kit could cover. A large eDNA surveys
usually consisted of several temporal times (Dejean et al.,2011; Turner et al., 2014; Bista et al., 2017), and dozens
even hundreds of spatial sampling sites (Takahara et al.,2012; Yamamoto et al., 2016), and sometimes each site
was sampled at two different section (surface and bottom)(Yamamoto et al., 2016). As a result, a lot of money was
spent in eDNA extraction part. Cheaper and efficientmethod of extracting eDNA of macro-organisms wastherefore a significative step in eDNA surveys.
Here, we examined certain cheaper kits, AxyPrep
DNA Gel Extraction Kit (Axygen) and Tissue DNA Kit(Omega bio-tek) as the potential DNeasy Blood andTissue Kit replacer. We used these kits to extract eDNA
from filter membranes after filtration. To better reveal
the effectiveness of three kits, digital PCR (dPCR) wasused by figure out the number of Sepiella japonica DNA
copies. The reason why we chose Sepiella japonica as
our target species was that although this species has
important economic value and was suffering severe
resource declining, there was no eDNA study of Sepiellajaponica. More sensitive and convenient eDNA resource
surveys of Sepiella japonica are necessary to be carriedout in the future. In present study, both field and indoors
water samples were tentatively collected. Because fieldwater was complicated and there was a lot of uncertainty
of eDNA distribution (Roussel et al., 2015), sometimes
specific-target digital PCR (dPCR) of field water might
can’t be put into effect. If dPCR results revealed that there

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was no eDNA copy of Sepiella japonica, we would use
nonspecific-target agarose electrophoresis to measure thetotal eDNA of field water samples. Sediment concentration
had a severe influence on eDNA yield and varied
significantly between water samples (Ficetola et al., 2008;
Thomsen and Willerslev, 2015). So, we simultaneously
measured the sediment concentration of water samples todetermine our experiment. We hope this kits’ comparison
result would cut down the extracting cost and contribute to
the subsequent eDNA surveys in the future.

MATERIALS AND METHODS

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Species-specific primers and probe design
Cytochrome c oxidase I (COI) sequences of Sepiellajaponica as well as all Cephalopoda species existing in
the coastal waters of Zhoushan were downloaded from
Genbank. To give consideration to both the efficiency andspecies-specificity of primers and probe, we constantly
adjusted the parameters (position, length, Tm and soon) of Primer Express 3.0.1 software (Thermo Fisher
Scientific, Waltham, MA). Ultimately, the sequences ofthe designed specific primers and probe were listed asfollows: F-2317: 5’-CACCAGACATAGCCTTCC-3’;
R-2471: 5’-GCCAGCATGAGATAGATTAC-3’; andPro-2432: 5’-HEX-TGTTCATCCAGTTCCAGCACCTTAMRA-3’. To check the specificity of the primers andprobe, we performed digital PCR separately by using the
DNA of Octopus variabilis, Octopus ocellatus, Sepia
esculenta, Sepiella japonica, Loligo kobiensis, Sepiolabirostrata and Loligo beka (the common species in
Zhoushan coastal waters). The digital PCR conditions
were described in section 2.5. The digital PCR results
confirmed their species-specification.
Water sampling and filtration
Water sampling was conducted in Zhoushan City,
Zhejiang Province, because its close distance to laboratory
could benefit eDNA transportation, preservation andsubsequently filtration. In consideration of difference
between field and indoor water environment, both field andindoors studies were carried out. Exactly, four nonspecifictarget water sampling sites (not sure whether there was
Sepiella japonica or not) were set throughout the field
river (sample 1 to sample 2) and sea (sample 3 to sample
4) and one water sampling sites (sample 5) speciallytargeting at Sepiella japonica was set at a breeding base(located in Xixuan island, a professional Sepiella japonica
farm) (Fig. 1). The water sampling started at April 17,
2017 and ended at the same day. Briefly, water samples
were collected from the water surface using a bucket andthen parallelly divided into 12 plastic cups (three kits

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Method of Extracting Environmental DNA from Filter Membranes

group × triplicate at each site, and the rest three wereused to measure sediment concentration of every sample(in section 2.3); volume of all plastic cup was 1.5 L). To
minimize cross-contamination, the bucket was bleachedusing 0.1% sodium hypochlorite for about ten minutes
before sampling and washed twice or more with surface
water at each sampling site and was then placed in water forseveral minutes before each water collection (Yamamoto
et al., 2017). All water samples were simply added thechemicals benzalkonium chloride, at a final concentrationof 0.01% to suppress the degradation of eDNA and
immediately transported to our laboratory (Yamanaka etal., 2017). All water samples were filtered through WCN
filters (Whatman Cellulose Nitrate Membrane Filters,
7184-004) of 0.45 μm average pore size. Two sets offiltration equipment (Combisart 3-branch stainless steel
manifold, GM-0.33A pump) were used to accelerate theprocess of experiment. A total of 300 ml of every sample
was uniformly filtered because later blocking made somesamples cannot be filtered more. The filter funnels andmeasuring cups used for filtration were also bleached after
filtration using 0.1% sodium hypochlorite. In addition,
to verify the effectiveness of the bleaching, we filteredartificial seawater with every filter funnel and measuring
cup at every site (equipment negative control) (Yamamoto
et al., 2016).

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2.2. Then, these filter membranes were dried completely
in an electro-thermostatic blast oven (Shanghai Hasuc
Instrument Manufacture Co., Ltd. DGH-9055A) at 56°C.
The above electro-thermostatic blast oven measured
these filter membranes again to determine the sedimentconcentration of water samples.
DNA extraction
In this step, filter samples were subjected to DNA
extraction following the method referring to Miya et al.
(2015), the specification of AxyPrep DNA Gel Extraction
Kit and Tissue DNA Kit, respectively. Briefly, each filter
was rolled into a cylindrical shape using sterile forceps
and placed into a spin column (EZ-10 Spin Column &Collection Tube; Bio Basic Inc., Ontario, Canada). The
silica membranes of the columns were removed before
use. The columns were centrifuged at 6000g for 1 minand excess water on the filter recovered as the filtrate wasdiscarded. As for DNeasy Blood and Tissue Kit, AxyPrep
DNA Gel Extraction Kit, and Tissue DNA Kit, the reagent
names and dosage of each step were listed, respectively
as follows (three brackets in following steps correspondedwith three kits):
1: (200µl ultrapure water, 200 µl buffer AL and 10 µl
proteinase K) or (400 µl buffer DE-A and 10 µl proteinaseK) or (200 µl ultrapure water, 200 µl buffer TL and 10 µl
proteinase K) were dispensed onto the filter, and the spincolumns were then incubated at 56°C for 30 min.
2: The spin columns were centrifuged for 1 min at
6000g to elute the filtrate, and this elution was moved to
new 1.5-ml micro-tubes.
3: Then, 400 µl of TE buffer was added to eachfilter and incubated for 1 min at room temperature. Spincolumns were centrifuged for 1 min at 6000g to recoverany DNA remaining on the filters. The first elutions werethen returned to the 2-ml collection tubes containing thesecond elutions.
4: Then, 200 µl (buffer AL) or (buffer DE-B) or (buffer

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Measurements of sediment concentration
Sediment concentration had a severe influence on
eDNA yield (Turner et al., 2014), and in whole coastal of
Zhoushan, sediment concentration was high everywhere.
Thus it was necessary to consider the influence of sedimentconcentration. Before extraction, dry weight of each filter
membrane was determined by a SECURA225D-1CN
electronic balance (Sartorius Mechatronics, weighing
range: 0-120g; degree of accuracy: 0.01mg). Afterwards,
the water samples targeting at sediment concentration
measurements were filtrated as those described in section

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Fig. 1. Water sampling sites of this study. From left to right, the map was gradually enlarged.

Z. Chen et al.

4BL) and 610 µl ethanol were added to each collection
tube and mixed well by pipetting. eDNA in each solution
was collected and purified using the three different kits bycentrifuging the solution by using the provided respective
spin columns in three stages because of the large volumeof each filtrate.
5: The silica-gel membrane was washed two times
using washing buffers (AW1 and AW2) or (W1 andW2) or (HBC and DNA Wash buffer), according to the
manufacturer’s instructions.
6: DNA was eluted from the spin columns with 100 µlof the provided (AE) or (Eluent) or (Elution) buffer.
PCR and digital PCR (dPCR)
We performed dPCR by using the Sepiella japonica
primers and probe. The detail of dPCR was followed themethod referring to Doi et al. (2015a, b). Each dPCR
reaction mixture (20 µl) contained 2 µl of DNA template,
900 nM of each primer, and 125 nM TaqMan probe in a
1 × Bio-Rad Supermix (Bio-Rad, Hercules, CA, USA),
which was mixed with Bio-Rad droplet generator oil andpartitioned into 15,000–20,000 droplets by using the BioRad QX-100 droplet generator (Bio-Rad). The droplets ofindividual samples were separately applied to each well ofa 96-well PCR reaction plate. PCR was performed in the
96-well plate sealed with pierceable sealing foil by using
the Gene Amp 9700 thermocycler (Life Technologies).
For dPCR analysis, triplicated PCR negative controls
were used in which 1 µl ultrapure water was added to eachreaction instead of the eDNA template. PCR conditions
were 10 min at 95°C, 40 cycles of denaturation for 30 s
at 95°C and extension for 60 s at 60°C with ramp rate of
2.5°C/s, followed by 10 min at 95°C and a hold at 4°C.
After PCR amplification, the plate was transferred to the
Bio-Rad QX-100 droplet reader (Bio-Rad). Each droplet
in the well was checked for TaqMan fluorescence to countthe number of droplets that yielded positive/negative
results. We used Bio-Rad’s Quanta Soft software version
1.3.2.0 to quantify the copies of target DNA. Threshold fora positive signal was determined according to the QuantaSoft instructions. Any droplet beyond the fluorescencethreshold was counted as a positive event. We used themean and standard deviation values for statistical analyses.
At the beginning of our study, we intended touniformly use eDNA copies of Sepiella japonica in bothfield and indoors water samples to evaluate the extraction
efficiency of three kits. However, in whole coastal of
Zhoushan, sediment concentration was high everywhere
and resulted in rare marine-life survival (Jiang and Guo,2016). Some previous experimental results also revealed
no Sepiella japonica inhabiting in these field waters. Thus
if dPCR results revealed that there was no eDNA copy

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of Sepiella japonica, we would use nonspecific-target
agarose electrophoresis to measure the total eDNA offield water samples (nonspecific-target meant much moretotal eDNA could be amplified and would show a moremanifesting result). A professional set of universal PCR
primers (12S rRNA) for metabarcoding environmental
DNA from fishes was used to analyze these 4 field water
samples by seeing their agarose gel. The sequence of thisuniversal PCR primers used in this study were: MiFish-U-F(5’-GTCGGTAAAACTCGTGCCAGC-3’) and MiFishU-R (5’-CATAGTGGGGTATCTAATCCCAGTTTG-3’).
These two primers were professionally designed for eDNAof teleost fishes (Miya et al., 2015). PCR was carried out
in a 25 µl reaction mix containing DNA template (1 µl,
50 ng/µl), forward primer (MiFish-U-F, 1 µl, 10 µM/L),
reverse primer (MiFish-U-R, 1 µl, 10 µM/L), dNTPs (2μl, 2.5 mM/L each), EasyTaq DNA Polymerase (0.15µl, 5 U/µl) and 10 × PCR buffer (2.5 µl, 25 µM/L). A
Biometra thermal cycler (Gottingen, Germany) with thefollowing given procedure: one initial denaturation (95°C,
3 min), 35 cycles consisting of denaturation (94°C, 20 s),
annealing (50°C, 20 s) and extension (72°C, 20 s), and one
final extension (72°C, 3 min), was employed to put PCR
amplification into effect. All 12 DNA template samples
extracted from 4 field water samples were run in duplicate.
Triplicated PCR negative controls were also used in which
1 µl ultrapure water was added to each reaction insteadof the eDNA template. 1% agarose gel was employed tocheck the existence of target fragments. DNA bands werevisualized by staining with ethidium bromide.

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Statistical analysis
All data on sediment concentration and DNA yield
were calculated as milligram (mg) and DNA copies per
sample water volume, respectively, and used for statisticalanalyses. The average concentrations of sediment in fivewater samples and eDNA of Sepiella japonica in thethree kits groups were compared by analysis of variance(ANOVA) followed by the Tukey–Kramer post hoc test
using R ver. 3.0.2 (R Core Team 2013). The minimum
level of significance was set at p ≤ 0.05.

RESULTS
Sediment concentration
The measured sediment concentrations of fivewater samples (1 to 5) were: 16.33 ± 2.57, 9.3 ± 1.88,
50.23 ± 3.27, 50.5 ± 1.97, and 27.63 ± 7.06 mg/300 ml
water (mean ± standard deviation), respectively (Fig. 2).
ANOVA followed by a post hoc test revealed no difference
between sample 3 and sample 4 (p = 0.959), but did reveal
a significant difference in all rest combinations (p < 0.001).

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Method of Extracting Environmental DNA from Filter Membranes

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Kit filtration group, the eDNA concentrations of AxyPrep
DNA Gel Extraction Kit and Tissue DNA Kit groups werereduced to 83% and 51%, respectively.

Fig. 2. Sediment concentration of all water samples.

DNA copies of water samples
Blank samples showed negative results for DNA
copies. Results of dPCR revealed that there was no S.
japonica DNA copy in the all four field water samples.
The average Sepiella japonica DNA concentration (mean± standard deviation) of indoors water samples (water
simple 5) extracted by DNeasy Blood and Tissue Kit,
AxyPrep DNA Gel Extraction Kit and Tissue DNA Kit
were (4.62 ± 0.72) × 105, (3.83 ± 0.56) × 105, and (2.35 ±0.27) × 105 copies/300 ml aquaculture water, respectively(Fig. 3). ANOVA analysis revealed no difference between
the DNeasy Blood and Tissue Kit and AxyPrep DNA
Gel Extraction Kit groups (p = 0.061), but did reveal a
difference between the DNeasy Blood and Tissue Kit andTissue DNA Kit groups (p < 0.001), and between AxyPrep
DNA Gel Extraction Kit and Tissue DNA Kit groups (p =0.032). In comparison with the DNeasy Blood and Tissue

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Fig. 3. The DNA yield (copies/300 ml) of Sepiellajaponica extracted from aquaculture water (indoors water
sample). There was no difference in eDNA yield between
the DNeasy Blood and Tissue Kit and AxyPrep DNA Gel
Extraction Kit groups (p=0.061).

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Agarose gel electrophoresis
One amplification of water sample 1 extracted by
AxyPrep DNA Gel Extraction Kit failed. Others of water
sample 1 and sample 2 had a clear and bright major band.
Length of PCR products was about 220 bp, which wereconsistent with the description of this universally PCRprimers designed by Miya et al. (2015). Water sample 3
and 4 did not amplify at all in all three kits group. Gels in
Tissue DNA Kit group showed not only a major band but
also some obvious smear bands in the same lane.

Fig. 4. Agarose gel electrophoresis results of 4 field water samples. M, DNA marker.

Z. Chen et al.

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DISCUSSION
From the agarose gel image (Fig. 4), all three kitsobtained similarly bands. Apart one well of water sample
1 that was not amplified due to certain unknown reason,
when DNeasy Blood and Tissue Kit group showed clear
and bright major bands in water sample 1 and sample
2, AxyPrep DNA Gel Extraction Kit and Tissue DNA
Kit synchronously showed; when AxyPrep DNA Gel
Extraction Kit and Tissue DNA Kit could not extract
eDNA from water sample 3 and sample 4, DNeasy Bloodand Tissue Kit also failed in the same lane position. This
fact roughly but clearly suggested that AxyPrep DNA
Gel Extraction Kit and Tissue DNA Kit might be able toreplace DNeasy Blood and Tissue Kit as the method of
extracting environmental DNA from filter membranes.
More specifically, lane 1 to lane 12 of Tissue DNA Kit
group mingled with smear bands, but those of AxyPrep
DNA Gel Extraction Kit and DNeasy Blood and Tissue
Kit group didn’t, which indicated using Tissue DNA Kit to
extract eDNA might produce more impurities.
Sediment concentration had a severe influence on
eDNA yield, which was also been explored by Turner et al.
(Turner et al., 2014). Sampling site 3 and 4 were directly
located in the coast of open sea. A sturdy dam was built
along this coast and thus resulted in the sediment reductionof sample 1 and 2. Sediment concentration of sample 3
and sample 4 had no difference (p = 0.959), but was 3.08
and 5.41 times higher than sample 1 and sample 2 (p <0.001) (Fig. 2), respectively. High sediment concentration
made against fish survival (Watts et al., 2003), caused
much physical damage to DNA molecule and brought
about more extraction impurities. Such highly turbid water
also usually contained many PCR inhibition substances.
Some fisherman also caught fish by gill nets and cagenets in this area. It was confirmed that fish species existed
in the coast of this open sea. The 12S primers were
extremely universal as well, which were applicable for
70 families and 152 genera marine fishes at least (Miyaet al., 2015). Thus, although we did not further determine
what substances they are, it was reasonably inferred that
inhibition substances and extraction impurities cannot becompletely removed by three kits. As the final reflection of
low eDNA yield and high PCR inhibition, in agarose gel,no matter what kits were used to extract water sample 3 and4, no bands appeared in the lane position of these samples.
Although sample 1, 2 and 5 also had a heave weight, thecomponent of them might be organic substance rather thansediment. Because after filtration, color of filter membrane
was sandy brown of sample 3 and 4, but slight greenishyellow of sample 1 and 2 and light black of sample 5.
The digital PCR study suggested that water samples

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for eDNA analyses could be optionally extracted fromfilter membranes by using AxyPrep DNA Gel Extraction
Kit. On average, DNeasy Blood and Tissue Kit appeared
to be the most effective and Tissue DNA Kit was the worst.
In digital PCR experiment of Sepiella japonica, which
focused on DNA copies, reductions in DNA concentration
were both observed in AxyPrep DNA Gel Extraction
Kit and Tissue DNA Kit group comparing to DNeasy
Blood and Tissue Kit. This revealed that the chemicalcomponents and physic material might exist somedifference in these three kits, and such difference caused
the extraction effectiveness variation. However, all threekits were effective in extracting DNA of water samples, asconfirmed by the results of PCR and digital PCR. Although
the yield of eDNA extracted by DNeasy Blood and Tissue
Kit was 1.21 times higher than that extracted by AxyPrep
DNA Gel Extraction Kit, no difference between these twogroups (p = 0.061) was revealed.
Different from the popular and thoroughly developed
use of DNeasy Blood and Tissue Kit (Thomsen et al.,2012; Takahara et al., 2013; Miya et al., 2015; Doi etal., 2017; Minamoto et al., 2017), as far as we know, nopeople before us used AxyPrep DNA Gel Extraction Kit tocarry out the eDNA extraction of macro-organisms. The
operation procedure of AxyPrep DNA Gel Extraction Kit
could promisingly improve in the future. As some studies
had revealed, the length of eDNA dissolved in waters waspretty incomplete and short (Deiner et al., 2015; Jo et al.,2017; Tsuji et al., 2017). AxyPrep DNA Gel Extraction
Kit also specifically noted that if the DNA fragment was <400 bp, isopropanol should also be added when extracting.
In our present studies, we did not use isopropanol, whichmight pull down the performance of AxyPrep DNA Gel
Extraction Kit. Besides, the incubation object of AxyPrep
DNA Gel Extraction Kit was mainly agarose (saccharides),whereas DNA molecule was most wrapped in biologicalmembrane of cells, organelles or tissue fragments
consisting of lipid, protein and so on (Deiner et al., 2015;
Tsuji et al., 2017). Unified 10 ul proteinase K in AxyPrep
DNA Gel Extraction Kit might was not enough to incubate
biological membrane. Because as the name suggested,
DNeasy Blood and Tissue Kit itself had tissue incubation
ability but AxyPrep DNA Gel Extraction Kit didn’t. The
extraction efficiency of DNA from filter samples might be
improved by adding more proteinase K.
Based on the efficiency and promising improvementin the future, the price of kits was a significant factor
affecting kit selection in eDNA studies. A large scale
eDNA surveys usually consisted of several temporal times(Dejean et al., 2011; Turner et al., 2014; Bista et al., 2017),
dozens even hundreds of spatial sampling sites (Takahara
et al., 2012; Yamamoto et al., 2016), and each site was

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Method of Extracting Environmental DNA from Filter Membranes

also sampled at two different section (surface and bottom
waters) (Yamamoto et al., 2016). Sometimes, triplicate
was also done at every sample site (Yamamoto et al.,2016). Thus, such a survey need hundreds to thousands
filter membranes to filtrate. The price of DNeasy Bloodand Tissue Kit (50 times), AxyPrep DNA Gel Extraction
Kit (250 times) and Tissue DNA Kit (50 times) was
respectively 2000 RMB, 750 RMB and 550 RMB.
Attention should also be paid to DNeasy Blood and Tissue
Kit, because double dosage of buffer AL was used when
extracting (see section 2.5, or Takahara et al., 2012; Miyaet al., 2015; Yamamoto et al., 2016; Minamoto et al., 2017
and so on). Thus the average price of one filter membrane
extraction was respectively 80 RMB, 3 RMB and 11 RMB.
Tissue DNA Kit had the worst eDNA extraction efficiency,
its price was also disadvantaged. DNeasy Blood and Tissue
Kit was the most effective kit, but it was too expensive. In
small-scale eDNA studies, DNeasy Blood and Tissue Kit
might be best; but in large scale eDNA surveys, combined
the extraction efficiency, improvement prospect and use
cost, we thought AxyPrep DNA Gel Extraction Kit wasalso a good choice.

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AxyPrep DNA Gel Extraction Kit as an substitutefor DNeasy Blood and Tissue Kit could remarkably cut
the extracting cost in eDNA surveys. AxyPrep DNA Gel
Extraction Kit might be an optional low-cost method of
extracting environmental DNA of macro-organisms fromfilter membranes in large scale eDNA surveys.

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ACKNOWLEDGMENTSOThis work was supported by the National Key Researchand Development Program of China (2019YFD0901301),
National Science Foundation of China (41776171,
41806180) and the Scientific Research Foundation of
Hainan Tropical Ocean University (No. RHDRC201907).
Statement of conflict of interest
The authors have declared no conflict of interest.

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