CN107099595A - Fish natural propagation monitoring method based on environment DNA technology - Google Patents
Fish natural propagation monitoring method based on environment DNA technology Download PDFInfo
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- CN107099595A CN107099595A CN201710334341.6A CN201710334341A CN107099595A CN 107099595 A CN107099595 A CN 107099595A CN 201710334341 A CN201710334341 A CN 201710334341A CN 107099595 A CN107099595 A CN 107099595A
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Abstract
The present invention relates to a kind of method judged based on environment DNA technology fish natural propagation behavior, including：Water sampling, DNA are extracted, purpose fragment ddPCR is expanded and concentration analysis.The present invention is based on environment DNA identification technology, fished for independent of traditional ovum seedling, eat ovum fish anatomy, sampled point collection water sample only need to be rationally designed, then it is that can determine whether whether target species occurs oviposition behavior and determine spawning ground position that concentration analysis is carried out to target species.On the one hand the stock of fish is protected to greatest extent, on the other hand avoid traditional fishing operation and expend the inconvenience that the restrictive factor such as time length and the more difficult capture of part fingerling is brought.
Description
Technical field
The present invention relates to Molecular Ecology Techniques field, the fingerling concentration analysis of water body environment DNA sample is refered in particular to, is referred specifically to
A kind of fish natural propagation monitoring method based on environment DNA technology.
Background technology
Due to the reason such as hydraulic engineering construction, environmental pollution and transition, overfishing, in the natural water such as the Yangtze river basin,
Fish population quantity and diversity are successively decreased year by year, and the construction of dam has blocked migration fishes passage, such as mandarin sturgeon is due to Pueraria lobota
The foundation of continent dam engineering can not be traced back to the breeding of original spawning ground, in Ge Zhou Ba new spawning ground formed below.Such as reservoir area
The Fish Community Structures that setting up makes script flowing water type fish account for main body are slowly evolved into hydrostatic fish structure composition in the majority.Root
After finding that Three Gorges Dam is set up according to monitoring for many years, the quantity of four large Chinese carps falls sharply, in order to avoid dam is produced not to chinese carp
Good influence, Three Gorges group has organized the ecological dispatching bred for four large Chinese carps, based on ovum seedling resource quantity monitoring method,
It is aided with reproductive population Monitoring of Quantity and hydrologic monitoring, the tentative ecological dispatching for assessing Three Gorges Reservoir for chinese carp natural propagation is imitated
Really, experiment gives the factors such as water temperature, the continuous dayses that rise, crest discharge, initial flow, flow amount of increase and chinese carp oviposition behavior
Between relation.And by way of erect-position and mobile monitoring point are fixed to river subsection setup and combines observation, according to fishing for
The fish-egg of different growing periods and the fry of early development, with reference to water temperature condition at that time, extrapolate needed for fish-egg (seedling) is developed
Time, the driftage of ovum (seedling) is then being extrapolated according to the section mean flow rate of river at that time；Sometimes also in relation with dissection parent population and food
Ovum fish, understands parent population gonad development situation and masses' knowhow judges the accurate position in some spawning ground.
Although the above method can monitor chinese carp oviposition behavior and positioning spawning ground scope, because different river discharges are poor
Different, hydrologic(al) regime differs, and the spawning ground river section calculated has larger uncertainty.And conventional method labor intensity is big,
Operation difficulty is high, so, it is very necessary to find a kind of method that can quickly, conveniently monitor fish natural propagation behavior.
Environment DNA (environment DNA), which refers to come off from organism, to be discharged into large natural environment (such as empty gas and water, soil
Earth) DNA, including the extracellular DNA molecular that dissociates after DNA in cell and clasmatosis.Environment DNA technology is exactly direct
DNA fragmentation is extracted from the environmental samples such as water, soil, deposit, then passes through PCR and sequencing equimolecular Biological Detection skill
Art carrys out qualitative or quantitative target product, so that it is determined that the research method of target organism distribution in this context and functional character.
Jessica etc. is studied to U.S. Grover Starling lake carp distribution situation and its environment DNA.Study on monitoring tight for many years makes
Its distribution and density case to carp in the lake is had at fingertips, taken in 22, lake place superficial water, subsurface water and
Deposit carries out carp DNA extractions, as a result shows, the environment DNA content of carp distribution high density area is high in water body example, on the contrary
It is as the same, and Sediment environment DNA is then unchanged.In addition, the DNA content of superficial water and subsurface water has no significant difference, i.e.,
The vertical characteristics of eDNA concentration are into uniform state, and the eDNA in the smaller spatial dimension of more than ten meters to hundreds of meters of distance objective kind
It is in irregular distribution in the horizontal direction that significant change from big to small, i.e. eDNA concentration, which occur, for concentration.In fact, eDNA is dense
Degree whether can accurate response go out sampled point Species structure situation and eDNA degraded it is closely related, different sampled points, sampling
Position is influenceed by many factors such as water quality, pH value, illumination, humus, chemical substances, and its degradation rate is different, detects
EDNA concentration distribution situations are also different.Show such as above-mentioned Jessica results of study, the eDNA concentration of fish high density area>It is low
The eDNA concentration of density region>The eDNA concentration of deposit, and in Pilliod research, the eDNA of different sampling location detections
Concentration value has no significant change.In recent years, environment DNA technology is widely used in fish species identification, diversity analysis and determined
In amount analysis.Water environment DNA gene identification method is inferring that water biological species are geographical for traditional direct observational method
It is more easy to obtain sample in distribution, and due to when small fragment DNA can may be present very long one section under drying, freezing or no light condition
Between, in the case that biological density is very low, environment DNA technology is still effective, and no matter water body state is fixed or flowed
Situation, this also cause environment DNA technology turn into Ecological Protection study in a very valuable instrument.
DdPCR refers to droplet digital pcr system, is the emerging round pcr of a class grown up in the recent period, the production of its fluorescence signal
Raw principle is identical with qPCR, and drop generator can receive the Fluorescence PCR system " segmentation " containing nucleic acid molecules into tens thousand of
The droplet of meter level, nucleic acid molecules are dispensed at random in droplet, each droplet or without nucleic acid target molecule to be detected, or containing extremely
A few nucleic acid target molecule to be checked, and each droplet is an independent PCR reactor.After being expanded through PCR, analyzer by
Individual that each droplet is detected, the droplet interpretation for having fluorescence signal is " 1 ", and the interpretation without fluorescence signal is " 0 ", so that will
Fluorescence analog signal digital, finally according to Poisson distribution principle and the ratio of positive droplet, analysis software can directly give and treat
Examine the copy Particle density of target molecule.So, standard curve need not be set up by carrying out Concentration Testing using ddPCR methods, greatly
Amount is it is demonstrated experimentally that ddPCR has more preferable accuracy compared to qPCR, particular for species concentration quantifying in the case of relatively low
Analysis.
The content of the invention
The purpose of the present invention be overcome above-mentioned conventional method it is not enough there is provided one kind independent of fingerling in itself, using fish
Chondrioid gene obtains target species DNA concentration situation of change by ddPCR analyses, so as to judge fish natural propagation behavior
Method.
To achieve these goals, the present invention provides a kind of fish natural propagation monitoring method based on environment DNA technology,
Comprise the following steps：
Step 1：According to research waters size, reasonable Arrangement sampled point gathers water sample.
Step 2：Above-mentioned water sample is stored refrigerated in 4 DEG C, and in 24h, suction filtration is carried out using the filter membrane in 0.22 μm of aperture,
And filter membrane is placed into -20 DEG C of degree preservations to DNA extractions.
Step 3：DNA genomes are extracted using water sample DNA extraction kit.The DNA extraction kit producer isWater DNA Kit(Omega)。
Step 4：Primer, probe validation checking are carried out to purpose fragment using qPCR, in experimental system, added relative
After the primer, probe, template DNA, rox reference fluorescents and the mix that answer volume, fluorescence is carried out together with negative control, positive control
Quantitative pcr amplification.Verify after primer, probe validity, carry out ddPCR.
Step 5：Analyzed by the sample collection of Consecutive Days, eDNA concentrations in the same sampled point monitoring time, tentatively
Judge whether target species occurs oviposition behavior；Compared by different sampled points with the eDNA concentrations of time, thus it is speculated that spawning ground
Position.
The collection water sample of the step 1, is the 1-2L water bodys obtained after different depth layering collection water sample is mixed.
The suction filtration of the step 2, in the case where concrete water quality is larger, can be carried out using multiple filter membranes will after suction filtration, suction filtration
Obtained filter membrane periphery rest is subtracted, to reduce the usage amount of reaction solution.
In the DNA extraction process of the step 3, every cutting edge filter membrane is cut into 4 area identicals sectors along center line
Afterwards, add in the centrifuge tube equipped with reaction solution, be placed in ultrasonically treated in ultrasonic instrument, and whole ultrasonic procedure enters to centrifuge tube
In the ranks break and be vortexed, to obtain the DNA on filter membrane to greatest extent, DNA extractions are then carried out according to kit step.Specifically
Step is：The filter membrane sheared is put into 50ml centrifuge tubes, adds reaction solution, be placed in KQ5200E type ultrasonic washing instrument devices
Progress is ultrasonically treated in (ultrasonic power 400w), according to specific adhesion situation not timing attachment is more in ultrasonic procedure
Filter-membrane centrifugal tube is placed on turbula shaker and is vortexed, to obtain the DNA on filter membrane to greatest extent, it is ultrasonically treated until
Attachment on filter membrane is fully entered in reaction solution, and whole ultrasonication is not heated.
In the step 4, qPCR reaction systems are 20 μ L, DNA profiling 2 μ L, primer, probe, rox each 0.4 μ L, Taq
Premix 10 μ L, ddH20 6.4μL；PCR amplification conditions are：94 DEG C of 3min pre-degenerations, 94 DEG C of 15s denaturation, 60 DEG C of 30s annealing
Extension, 40 circulations.DdPCR amplification reaction systems are 20 μ L, the wherein μ L of DNA profiling 2, the μ L of primer 1.8, probe 0.5 μ L, Taq
The μ L of 10 μ L, RNase/DNase-free water of Premix 3.9；By the 20 μ L PCR reaction solutions configured, droplet is transferred to
In generation card (DG8cartridge) sample holes, 70 μ L droplets oil is added into oil holes, QX2000 droplet types numeral is utilized
The drop generators of PCR instrument prepare reaction droplet；The reaction droplet prepared is transferred to correspondence in 96 hole PCR reaction plates respectively
Reacting hole in, sealed with aluminium film after (180 DEG C, 5sec), in the amplification of regular-PCR instrument, its amplification condition is：95 DEG C of 5min become in advance
Property, 95 DEG C of 30s denaturation, 60 DEG C of 1min annealing extensions, 40 circulations, 98 DEG C of 10min hold.
Beneficial effect of the present invention：
Chinese carp of the present invention can be firstly gathered at spawning ground scope, then treat the bar that suitably rises under suitable water temperature condition
Part issues production ovum, it is this naturally bunch, oviposition behavior can increase the chinese carp DNA being discharged into water body, this DNA concentration
Change be the present invention use technology to react chinese carp oviposition behavior with position spawning ground basis.Due to the production of chinese carp cluster
Ovum behavior only continues shorter a period of time, and main stream of the Yangtze water body flow is stronger, and flow field change is complicated, the scattered speed of chinese carp eDNA
The probability reduction that degree is comparatively fast collected, then chinese carp egg-laying time the adopting in real time as standard obtained using traditional monitoring for many years
Sample, the result error that can not only avoid DNA degradation from bringing, once and DNA can be detected in sample, theoretically
Can preferably the nearer population density of reaction distance sampled point the sampling time section distribution situation, i.e., the sampling time section eDNA it is dense
The high position of degree, species distribution density is larger, and vice versa.
1st, capture of this method independent of fingerling, only need in target water body certain limit, water sampling is carried out in the time
Acquisition target species information that can quickly, easy.For some rare Endemic fishs and the fingerling of more difficult capture, this method also compared with
It is more effective that tradition fishes for investigation.
2nd, quantitative analysis is carried out using ddPCR, without making standard curve, simplifies quantitative experiment operating procedure, and compare
In traditional qPCR, ddPCR has higher detection sensitivity, for some rare Endemic fishs in the case where concentration is very low
Concentration Testing can equally be carried out.
3rd, verified after primer, probe validity, water sample is expanded using ddPCR, each sample is not required to by qPCR
Replication.
4th, the DNA being discharged into due to chinese carp during laying eggs in water body, which is higher by, usually bunches when not laying eggs a lot, leads to
Crossing sample collection continuous many days in the same sampled point monitoring time, analysis eDNA concentrations can tentatively judge whether chinese carp sends out
Raw oviposition behavior.
5th, because chinese carp can bunch behavior before spawning in spawning ground scope, then spawning ground scope institute water sampling
Chinese carp eDNA concentration can be higher than other sampled points.Compared by different sampled points with the eDNA concentrations of time, production can be speculated
Ovum position.
Brief description of the drawings
Fig. 1 is to be of the invention in mausoleum mouth harbour to the rear kilometer range of river a small bay in a river about 36, and Three Gorges Reservoir is dispatched for 2016 for the first time
The period chinese carp eDNA average daily total concentrations of continuous 5 days；
Fig. 2 is to be of the invention in mausoleum mouth harbour to the rear kilometer range of river a small bay in a river about 36, and Three Gorges Reservoir is dispatched for 2016 for the first time
The chinese carp eDNA concentration of continuous 5 days of period each section；
Fig. 3 is of the invention in mausoleum mouth harbour to the rear kilometer range of river a small bay in a river about 36,2016 second scheduling of Three Gorges Reservoir
The period chinese carp eDNA average daily total concentrations of continuous 4 days；
Fig. 4 is of the invention in mausoleum mouth harbour to the rear kilometer range of river a small bay in a river about 36,2016 second scheduling of Three Gorges Reservoir
The chinese carp eDNA concentration of continuous 4 days of period each section；
The 6th large Chinese carp fish-egg variable density process of section (Yidu City) four during Fig. 5 monitors for the present invention；
The set website distribution of Fig. 6 safflowers；
The Monitoring Data of Fig. 7 safflowers set (removing H1) 11 websites.
Embodiment
The present invention is further illustrated with reference to embodiment, but the scope of protection of present invention is not limited to implement
The scope of example statement.
Embodiment 1
The natural propagation situation of (- 12 days on the 8th June in 2016) chinese carp when Three Gorges Reservoir is dispatched for 2016 for the first time.1st, sample
Product are gathered
According to conventional survey result for many years, in Yichang in Miao Zui harbours to the rear kilometer range river of river a small bay in a river about 36 section, 6 are set
Individual section gathers water sample, and each section sets the sampled point of left, center, right three, and each sampled point takes different depth water layer respectively
The water sample 2L mixed, and with being preserved in 4 DEG C of incubators.Sampled point particular geographic location is shown in Table 1.2nd, DNA is extracted
By above-mentioned water sample in 24h, it is filtered by vacuum by 0.22 μm of the fat-soluble filter membrane in aperture, according to multiple preliminary experiment ratio
Right, every liter of water body preferably uses 1 filter membrane, therefore filtered 2 filter membranes are put into 50ml centrifuge tubes into -20 DEG C and saved backup.
Using the water sample DNA extraction kit the of specialtyWater DNA Kit (Omega) are to above-mentioned filter membrane
Carry out DNA extractions.First filter membrane boundary part is cut before extracting, so that reaction solution fully soaks filter membrane, by every filter membrane
4 sectors are cut into along center line, are put into 50ml centrifuge tubes, 3ml SLX reaction solutions and 500mg micro glass pearls is added, are vortexed
3 minutes or uniformly it is covered with filter membrane to reaction solution, is put into Ultrasound Instrument and carries out ultrasonication, be during which interrupted vortex centrifugal pipe, so that
The DNA being stained with filter membrane is substantially dissolved in reaction solution, is carried out DNA extractions according to the step of kit specification afterwards, will be carried
DNA-20 DEG C taken is saved backup.
The specific address position of the different section sampled point of table 1
3rd, dd PCR are expanded
Designed primer, probe are subjected to validation verification on qPCR with the higher sample of fish body or concentration first
Afterwards.
Primer sequence is：SEQUENCE LISTING 1：
Both 2-F(qPCR)5’-GGCCGGAACAGGATGAACAGTT-3’
SEQUENCE LISTING 2：
Both 2-R(qPCR)5’-TAATAGTTGTGGTGATGAAGTTAATTG-3’
Probe sequence is：SEQUENCE LISTING 3：
Probe2 5’-FAM-CACGCAGGAGCATCCGTAGACCT-BHQ-3’
QPCR amplification reaction systems are 20 μ L, wherein DNA profiling 2 μ L, primer, probe, rox each 0.4 μ L, Tap
Premix10 μ L, ddH20 6.4μL；Amplification condition is：94 DEG C of 3min pre-degenerations, 94 DEG C of 15s denaturation, 60 DEG C of 30s annealing are prolonged
Stretch, 40 circulations.
DdPCR amplification reaction systems are 20 μ L, the wherein μ L of DNA profiling 2, the μ L of primer 1.8, probe 0.5 μ L, Tap
Premix 10μL,RNase/DNase-free water 3.9μL；Amplification condition is：95 DEG C of 5min pre-degenerations, 95 DEG C of 30s become
Property, 60 DEG C of 1min annealing extensions, 40 circulations, 98 DEG C of 10min hold.
The purpose fragment sequence of amplification is SEQUENCE LISTING 4：
ggccggaaca ggatgaacag tttacccgcc actcgcgggt aatcttgctc acgcaggagc
atccgtagac ctaacaattt tctccctcca cttagcaggt gtatcatcaa ttttaggggc
aattaacttc atcaccacaa ctatta
4th, concentration analysis
According to ddPCR experimental result, the target species copy number that each sample is included is learnt, 2 are shown in Table, its chinese carp calculated
Average daily eDNA total concentrations are shown in Fig. 1, and the chinese carp eDNA concentration of each section is shown in Fig. 2.Compare 6 section chinese carp eDNA in the monitoring time
Average daily concentration change situation, it is known that, during first time ecological dispatching the 5th day chinese carp environment DNA content occur it is a large amount of increase, with
EDNA concentration variance analysis a few days ago is in significant difference, and oviposition behavior occurs for chinese carp during judging this time to dispatch, and does not send out
The eDNA concentration radixes for producing chinese carp before ovum are smaller.Compare the chinese carp DNA concentration value of each section on the same day, it is known that Cmax
Value occurs in the 5th day the 4th section.In addition, each section DNA concentration value is it was found that fourth, fifth section is disconnected compared with other
Face concentration value is high, because water body flow direction is section one to section six, it is determined that section four is nearby spawning ground position.
The chinese carp DNA copy number that table 2 is detected during dispatching for the first time
Embodiment 2：The natural propagation of (- 22 days on the 19th June in 2016) chinese carp during 2016 second scheduling of Three Gorges Reservoir
Situation, method and step is identical with embodiment one, and table 3 is that the continuous time ddPCR quantitative analysis in 4 day of 6 sections, 18 sampled points is obtained
The chinese carp DNA copy number arrived, its average daily total concentration is shown in Fig. 3, and the concentration of each section is shown in Fig. 4.
The chinese carp DNA copy number detected during second of the scheduling of table 3.
According to ddPCR experimental result, the target species copy number that each sample is included is learnt, compares in the monitoring time 6
The average daily STb gene change in concentration situations of section chinese carp eDNA, it is known that, chinese carp environment DNA content during second ecological dispatching first day
It is maximum.Compare the chinese carp DNA concentration value of each section on the same day, it is known that Cmax value occur monitoring first day the 4th break
Face.Oviposition behavior occurs for chinese carp during judging this time to dispatch, and judges section four nearby as spawning ground position
It is inherent 6th during monitoring in order to which whether verification environment DNA methods can accurately judge fish reproduction behavior
Section is provided with traditional ovum seedling and fishes for a little daily 9 simultaneously:00,16:00 one top layer Jianggang net of placement takes ovum seedling about 1 hour, will
The ovum seedling collected carries out simple classification with size, transparency, color and luster, and the ovum seedling of doubtful chinese carp is taken back into indoor culture can extremely divide
The phase is distinguished, while recording the indexs such as flow, river flow velocity, the cross-sectional flow on the same day, is counted for ovum seedling density and stock number.Together
When, boating type detection is carried out in spawning ground using sonar detection equipment, judges that four large Chinese carps are numerous by the analysis to echo signal
Grow parent population quantity and signal distributions.
Early stage resource results collect four large Chinese carp fish-eggs, Yidu City's section fish-egg showing since 30 days Mays in 2016
Density and run-off daily process are shown in Fig. 5, it is known that 3 egg production peak times are occurred in that within 5,12 and 21 days in June, during spawning
Between be concentrated mainly on late May to June, July is then substantially without big ovum flood peak.Four gathered during eDNA monitorings are big
Chinese carp fish-egg density is as shown in table 4 with identical section eDNA concentration, by the correlation analysis between fish-egg density and eDNA concentration,
Understand, the eDNA concentration that fish-egg density and the section of the 6th section collection are detected is in extremely significantly correlated (P=0.0071<
0.01), illustrate that eDNA concentration can be very good to reflect the chinese carp Density Distribution situation in sampled point closer distance, high density area
EDNA concentration it is larger, the eDNA concentration of low density area is smaller.
Sonar surveillance website is arranged as shown in fig. 6, result is as shown in fig. 7, two websites of H2, H5 exist in safflower set website
Monitor a large amount of shoals of fish within 18-20 days, time (June 19) that relatively high DNA concentration value of this and environment DNA monitoring display occurs and
Place (the 4th and the 5th section) matches.
The concentration results that this method is detected according to ddPCR, judge twice dispatch during June 12, a left side on June 19
The right side there occurs chinese carp oviposition behavior, and tentatively judge that spawning ground is located near the 4th section.At the same time, from above-mentioned tradition
Ovum seedling fish in investigation, June 12, June 20 and 21 daily occur in that ovum flood peak during can learning scheduling twice；Separately
Outside, sonar walks boat detection and finds that family fish parent fish is more during dispatching is gathered in the 4th, near the 5th section, it was demonstrated that should
The validity of inventive method.So, in summary, the fingerling concentration monitoring method based on environment DNA can effectively react house
The natural propagation situation of fish.
Family's fish-egg seedling density-concentration value during the eDNA of table 4 is monitored
| Time | 6-8 | 6-9 | 6-10 | 6-11 | 6-12 | 6-19 | 6-20 | 6-21 | 6-22 |
| Ovum seedling density (A/m3) | 5.19 | 1.53 | 1.63 | 9.16 | 58.18 | 0 | 0.62 | 43.74 | 19.1 |
| EDNA concentration (copies/ μ l) | 108.7 | 38.7 | 21.9 | 71.3 | 5735.7 | 653 | 394.3 | 801.7 | 1677.3 |
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the embodiment with description.
。
SEQUENCE LISTING
<110>Yangtze River in China Three Gorges mandarin sturgeon research institute of group
<120>Fish natural propagation monitoring method based on environment DNA technology
<130> 2015
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Asian Carps
<400> 1
ggccggaaca ggatgaacag tt 22
<210> 2
<211> 27
<212> DNA
<213> Asian Carps
<400> 2
taatagttgt ggtgatgaag ttaattg 27
<210> 3
<211> 23
<212> DNA
<213> Asian Carps
<400> 3
cacgcaggag catccgtaga cct 23
<210> 4
<211> 146
<212> DNA
<213> Asian Carps
<400> 4
ggccggaaca ggatgaacag tttacccgcc actcgcgggt aatcttgctc acgcaggagc 60
atccgtagac ctaacaattt tctccctcca cttagcaggt gtatcatcaa ttttaggggc 120
aattaacttc atcaccacaa ctatta 146
Claims (6)
1. a kind of fish natural propagation monitoring method based on environment DNA technology, it is characterised in that：Described method includes：Water
Sample collection, DNA are extracted, purpose fragment ddPCR is expanded and concentration analysis, specifically include following steps：
Step 1：According to research waters size, reasonable Arrangement sampled point gathers water sample；
Step 2：By step 1 water sample in stored refrigerated, and within 24h, suction filtration is carried out using filter membrane, and filter membrane is placed -20
DEG C preserve to DNA extract；
Step 3：DNA genomes are extracted using water sample DNA extraction kit；
Step 4：Primer, probe validation checking are carried out to purpose fragment using qPCR, in reaction system, corresponding body is added
After long-pending primer, probe, template DNA, rox reference fluorescents and mix, fluorescent quantitation is carried out together with negative control, positive control
PCR is expanded, and after checking primer, probe validity, carries out ddPCR；
Step 5：Analyzed, tentatively sentenced by the sample collection of continuous several times, eDNA concentrations in the same sampled point monitoring time
Whether disconnected target species occurs oviposition behavior；Compared by different sampled points with the eDNA concentrations of time, thus it is speculated that spawning ground position
Put；
Complete the fish natural propagation monitoring based on environment DNA technology.
2. according to the method described in claim 1, it is characterised in that：The collection water sample of the step 1, is that different depth layering is adopted
The 1-2L water bodys obtained after collection water sample mixing.
3. according to the method described in claim 1, it is characterised in that：The suction filtration of the step 2, water sample can be entered using multiple filter membranes
Obtained filter membrane periphery rest is subtracted after row suction filtration, suction filtration；The filter sizes are 0.22 μm.
4. according to the method described in claim 1, it is characterised in that：In the DNA extraction process of the step 3, by every cutting edge
Filter membrane is cut into after 4 area identicals sectors along center line, is added in the centrifuge tube equipped with reaction solution, is placed in ultrasonic instrument
It is ultrasonically treated, and whole ultrasonic procedure carries out interruption vortex to centrifuge tube, to obtain the DNA on filter membrane to greatest extent, so
DNA extractions are carried out according to kit step afterwards.
5. method according to claim 4, it is characterised in that：In the ultrasonic power 350-450w, ultrasonication
Do not heat.
6. according to the method described in claim 1, it is characterised in that：In the step 4, qPCR reaction systems are 20 μ L, DNA moulds
Each 0.4 μ L, Taq Premix 10 μ L, ddH of plate 2 μ L, primer, probe, rox20 6.4μL；PCR amplification conditions are：94℃
3min pre-degenerations, 94 DEG C of 15s denaturation, 60 DEG C of 30s annealing extensions, 40 circulations；
DdPCR amplification reaction systems are 20 μ L, the wherein μ L of DNA profiling 2, the μ L of primer 1.8, the μ of 0.5 μ L, Taq Premix of probe 10
The μ L of L, RNase/DNase-free water 3.9；By the 20 μ L PCR reaction solutions configured, droplet card is transferred to（DG8
cartridge）In sample holes, 70 μ L droplets oil is added into oil holes, the micro- of QX2000 droplet type digital pcr instrument is utilized
Drip maker and prepare reaction droplet；The reaction droplet prepared is transferred to corresponding reacting hole in 96 hole PCR reaction plates respectively
In, sealed with aluminium film（180 DEG C, 5sec）Afterwards, expanded in regular-PCR instrument, its amplification condition is：95 DEG C of 5min pre-degenerations, 95 DEG C
30s is denatured, 60 DEG C of 1min annealing extensions, 40 circulations, 98 DEG C of 10min hold.
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| CN111690725A (en) * | 2020-07-03 | 2020-09-22 | 中国水产科学研究院珠江水产研究所 | Fish channel passing effect monitoring and evaluating method based on environmental DNA |
| CN113215275B (en) * | 2021-05-13 | 2022-12-09 | 水利部中国科学院水工程生态研究所 | Acipenser sinensis environment DNA digital PCR detection method |
| CN113215275A (en) * | 2021-05-13 | 2021-08-06 | 水利部中国科学院水工程生态研究所 | Chinese sturgeon environment DNA digital PCR detection method |
| CN113832238A (en) * | 2021-10-13 | 2021-12-24 | 中国科学院水生生物研究所 | Primer and probe for detecting environmental DNA of Lasa naked nojirimus fish and application of primer and probe |
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