CN115678963A - eDNA sampling experiment analysis method for water environment - Google Patents
eDNA sampling experiment analysis method for water environment Download PDFInfo
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- CN115678963A CN115678963A CN202211384836.7A CN202211384836A CN115678963A CN 115678963 A CN115678963 A CN 115678963A CN 202211384836 A CN202211384836 A CN 202211384836A CN 115678963 A CN115678963 A CN 115678963A
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Abstract
The invention discloses an eDNA sampling experiment analysis method for a water environment, which specifically comprises the following steps: s1, selecting an eDNA filter compatible with any suction pump; s2, standardization treatment, S3, environmental DNA extraction, S4, PCR amplification homogenization treatment, S5, sequencing treatment, and the invention relates to the technical field of water environment eDNA analysis. The method for water environment eDNA sampling experiment analysis can realize that the collected experimental sample can be well preserved aseptically by adopting the filtering membrane supported by the biodegradable hydrophilic material and matching with good standardized treatment, thereby well achieving the purpose of quickly and safely preserving the collected experimental sample, avoiding the occurrence of serious pollution of the collected experimental sample due to nonstandard sampling and preservation in the water environment eDNA sampling experiment analysis process, ensuring more accurate experimental analysis result and greatly facilitating the analysis work of experimenters.
Description
Technical Field
The invention relates to the technical field of water environment eDNA analysis, in particular to an eDNA sampling experiment analysis method for a water environment.
Background
Environmental DNA (eDNA) refers to the sum of DNA fragments that can be directly extracted from environmental samples (e.g., water, soil, air, ice core, etc.). The eDNA technology is a new biological investigation method technology in recent years and consists of 3 parts including eDNA acquisition, gene analysis and result analysis. Compared with the traditional method, the eDNA technology has the advantages of high sensitivity, time and labor saving, no damage to the investigated object and the like, and does not require the investigator to have the traditional biological identification and identification experience. At present, the eDNA technology is applied to the detection of the existence of target species (such as invasive species, endangered species and other rare species), the estimation of biomass, and the wide application prospect in the protection of aquatic ecosystems, and the purpose of analyzing the eDNA is to obtain the taxonomic information and the gene function information of the species to which the DNA belongs in the environmental samples. To put it more colloquially, scientists extract eDNA from environmental samples in order to analyze which species these DNAs belong to, respectively, and thus verify which species are present in the corresponding environment. The purpose of eDNA is in agreement with traditional animal and plant classification surveys, the first category of eDNA is similar to the environmental biodiversity survey by detecting as many DNA sequences as possible in environmental samples, analyzing the species classification information to which they belong in comparison with databases, and finally identifying all species living in this environment, this method is also called DNA macro-barcode (DNA polymorphism). In sample processing, DNA macros-codes were sequenced using either the common Polymerase Chain Reaction (PCR) or direct shotgun (shotgun) method, both of which are well established and allow for the convenient availability of multiple DNA sequences.
At present in the experimental analysis process of sampling to water environment eDNA, because the sampling is preserved and is not standard, make the experimental sample of gathering pollute seriously to lead to there being the increase error in experimental analysis result, can not realize through the filtration membrane that adopts biodegradable hydrophilic material to support and cooperate fine standardized processing, make the experimental sample of gathering can carry out fine aseptic save, can't reach not only fast but also safe carry out the purpose of preserving to the experimental sample of gathering, thereby bring very big inconvenience for experimenter's analytic work.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a water environment eDNA sampling experiment analysis method, which solves the problems that in the existing water environment eDNA sampling experiment analysis process, due to the fact that sampling and storage are not standard, the acquired experiment sample is seriously polluted, so that an experiment analysis result has increased error, and the acquired experiment sample can be well and aseptically stored and cannot be rapidly and safely stored by adopting a filtering membrane supported by a biodegradable hydrophilic material and matching with good standardized treatment.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: an eDNA sampling experiment analysis method for a water environment specifically comprises the following steps:
s1, selecting an eDNA filter compatible with any suction pump;
s2, standardization treatment: collecting 90-100L of water samples from a sampling place, filtering the sampled water by using the eDNA sampler in the step S1, independently packaging a filter, pre-installing a filter membrane, standardizing the filtration parameters of the eDNA samplers of all the samples, and storing after the standardization treatment is finished;
s3, extracting environmental DNA: transferring the standard-treated experimental sample obtained in the step S2 to DNA extraction equipment, adding a lytic enzyme, mixing and reacting for 2-3h, and performing centrifugal separation treatment through a centrifugal mechanism to obtain target DNA required by an experiment;
s4, PCR amplification homogenization treatment: synthesizing a primer joint by genetic engineering design according to the target DNA sequence obtained in the step S3, carrying out PCR amplification treatment on the designed primer joint by PCR amplification equipment, purifying PCR amplification products, and then carrying out quantitative homogenization treatment on the purified PCR products:
s5, sequencing: and (5) carrying out DNA sequence sequencing on the PCR product subjected to the quantitative homogenization treatment in the step (S4), constructing a DNA sequence library, and then generating a DNA sequence library report.
Preferably, the filter membrane of the eDNA filter in step S2 is composed of a biodegradable hydrophilic material, which can automatically preserve the captured eDNA by drying, without a filter membrane transfer step, without chemical or refrigeration.
Preferably, in the step S4, all samples need to be treated by the onesteptmmpcr inhibitor removal kit during the PCR amplification process, so as to eliminate the PCR amplification inhibition effect in the samples.
Preferably, the filtering membrane is composed of one or more of polyether sulfone, polylactic acid, polyhydroxybutyrate, polyvinyl alcohol, polycaprolactone or polybutylene succinate.
Preferably, the thickness of the filtering membrane in the step S2 is 1-1.2 μm, and the diameter is 45-50mm.
Preferably, the filtration parameters on the eDNA samplers for all samples are normalized in step S2 at a flow rate of 0.5-1.5L/min, a pressure threshold of 8-12psi, and a target volume of 0.3-0.7L.
Preferably, the lytic enzymes in step S3 include DNA helicase, DNA gyrase, DNA primase and DNA synthetase.
(III) advantageous effects
The invention provides an eDNA sampling experiment analysis method for a water environment. Compared with the prior art, the method has the following beneficial effects: the method for analyzing the water environment eDNA sampling experiment specifically comprises the following steps: s1, selecting an eDNA filter compatible with any suction pump; s2, standardization treatment: collecting 90-100L of water samples from a sampling place, filtering the sampled water by using the eDNA sampler in the step S1, independently packaging the filter and pre-installing the filter membrane, standardizing the filtration parameters of the eDNA samplers of all samples, and storing after the standardization treatment is finished; s3, extracting environmental DNA: transferring the standardized experimental sample obtained in the step S2 to DNA extraction equipment, adding a lytic enzyme, carrying out mixed reaction for 2-3h, and carrying out centrifugal separation treatment through a centrifugal mechanism to obtain the target DNA required by the experiment; s4, PCR amplification homogenization treatment: synthesizing a primer joint by genetic engineering design according to the target DNA sequence obtained in the step S3, carrying out PCR amplification treatment on the designed primer joint by PCR amplification equipment, purifying PCR amplification products, and then carrying out quantitative homogenization treatment on the purified PCR products: s5, sequencing: DNA sequence sequencing is carried out on the PCR product subjected to quantitative homogenization treatment in the step S4, a DNA sequence library is constructed, and then a DNA sequence library report is generated, so that the filtering membrane supported by a biodegradable hydrophilic material can be matched with good standardization treatment, the collected experimental sample can be stored in a good sterile manner, the purpose of storing the collected experimental sample quickly and safely is well achieved, the situation that the collected experimental sample is seriously polluted due to the fact that sampling and storing are not standard in the water environment eDNA sampling experimental analysis process is avoided, the experimental analysis result is ensured to be more accurate, and the analysis work of experimenters is greatly facilitated.
Drawings
FIG. 1 is a flow chart of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, the embodiment of the present invention provides four technical solutions: an eDNA sampling experiment analysis method for a water environment specifically comprises the following embodiments:
example 1
An eDNA sampling experiment analysis method for a water environment specifically comprises the following steps:
s1, selecting an eDNA filter compatible with any suction pump;
s2, standardization treatment: collecting 95L of water samples from a sampling place, filtering the sampled water by using the eDNA sampler in the step S1, independently packaging the filter and pre-installing a filter membrane, standardizing the filtering parameters on the eDNA samplers of all the samples, and storing after the standardization treatment is finished, wherein the filter membrane of the eDNA filter is made of biodegradable hydrophilic materials, can automatically store the captured eDNA by drying, does not need a filter membrane transfer step, does not need chemical or refrigeration, and is made of polyether sulfone and polylactic acid, the thickness of the filter membrane is 1.1 mu m, the diameter of the filter membrane is 47mm, and the filtering parameters on the eDNA samplers of all the samples are standardized under the conditions of flow rate of 1L/min, pressure threshold of 10psi and target volume of 0.5L;
s3, extracting environmental DNA: transferring the standard-treated experimental sample obtained in the step S2 to DNA extraction equipment, adding a lytic enzyme, mixing and reacting for 2.5 hours, and performing centrifugal separation treatment through a centrifugal mechanism to obtain target DNA required by the experiment, wherein the lytic enzyme comprises DNA helicase, DNA gyrase, DNA primer enzyme and DNA synthetase;
s4, PCR amplification homogenization treatment: synthesizing a primer joint by genetic engineering design according to the target DNA sequence obtained in the step S3, carrying out PCR amplification treatment on the designed primer joint through PCR amplification equipment, purifying PCR amplification products, carrying out quantitative homogenization treatment on the purified PCR products, and treating all samples by using an OnESTepTMPCR inhibitor removal kit in the PCR amplification process to eliminate PCR amplification inhibition in the samples:
s5, sequencing: and (5) carrying out DNA sequence sequencing on the PCR product subjected to the quantitative homogenization treatment in the step (S4), constructing a DNA sequence library, and then generating a DNA sequence library report.
Example 2
An eDNA sampling experiment analysis method for a water environment specifically comprises the following steps:
s1, selecting an eDNA filter compatible with any suction pump;
s2, standardization treatment: collecting 90L of water samples from a sampling place, filtering the sampled water by using the eDNA sampler in the step S1, wherein the filter is separately packaged and pre-loaded with a filter membrane, the filter parameters of the eDNA samplers of all the samples are standardized, the eDNA filter is stored after the standardization treatment is finished, the filter membrane of the eDNA filter is made of biodegradable hydrophilic materials, the captured eDNA can be automatically stored by drying, the filter membrane transfer step is omitted, chemical or refrigeration is not needed, the filter membrane is made of polyhydroxybutyrate and polyvinyl alcohol, the thickness of the filter membrane is 1 mu m, the diameter of the filter membrane is 45mm, and the filter parameters of the eDNA samplers of all the samples are standardized under the conditions of flow rate of 0.5L/min, pressure threshold of 8psi and target volume of 0.3L;
s3, extracting environmental DNA: transferring the standardized experimental sample obtained in the step S2 to DNA extraction equipment, adding lytic enzyme, mixing and reacting for 2h, and performing centrifugal separation treatment through a centrifugal mechanism to obtain target DNA required by the experiment, wherein the lytic enzyme comprises DNA helicase, DNA gyrase, DNA primer enzyme and DNA synthetase;
s4, PCR amplification homogenization treatment: synthesizing a primer joint by genetic engineering design according to the target DNA sequence obtained in the step S3, carrying out PCR amplification treatment on the designed primer joint through PCR amplification equipment, purifying PCR amplification products, carrying out quantitative homogenization treatment on the purified PCR products, and treating all samples by using an OnESTepTMPCR inhibitor removal kit in the PCR amplification process to eliminate PCR amplification inhibition in the samples:
s5, sequencing: and (5) carrying out DNA sequence sequencing on the PCR product subjected to the quantitative homogenization treatment in the step (S4), constructing a DNA sequence library, and then generating a DNA sequence library report.
Example 3
An eDNA sampling experiment analysis method for a water environment specifically comprises the following steps:
s1, selecting an eDNA filter compatible with any suction pump;
s2, standardization treatment: collecting 100L of water samples from a sampling place, filtering the sampled water by using the eDNA sampler in the step S1, independently packaging the filter and pre-installing a filter membrane, standardizing the filtering parameters on the eDNA samplers of all the samples, and storing after the standardization treatment is finished, wherein the filter membrane of the eDNA filter is made of biodegradable hydrophilic materials, can automatically store the captured eDNA by drying, does not have a filter membrane transfer step, does not need chemical or refrigeration, and is made of polycaprolactone and poly (butylene succinate), the thickness of the filter membrane is 1.2 mu m, the diameter of the filter membrane is 50mm, and the filtering parameters on the eDNA samplers of all the samples are standardized under the conditions of flow rate of 1.5L/min, pressure threshold of 12psi and target volume of 0.7L;
s3, extracting environmental DNA: transferring the standardized experimental sample obtained in the step S2 to DNA extraction equipment, adding lytic enzyme, mixing and reacting for 3h, and performing centrifugal separation treatment through a centrifugal mechanism to obtain target DNA required by the experiment, wherein the lytic enzyme comprises DNA helicase, DNA gyrase, DNA primer enzyme and DNA synthetase;
s4, PCR amplification homogenization treatment: synthesizing a primer joint by genetic engineering design according to the target DNA sequence obtained in the step S3, carrying out PCR amplification treatment on the designed primer joint through PCR amplification equipment, purifying PCR amplification products, carrying out quantitative homogenization treatment on the purified PCR products, and treating all samples by using an OnESTepTMPCR inhibitor removal kit in the PCR amplification process to eliminate PCR amplification inhibition in the samples:
s5, sequencing: and (5) carrying out DNA sequence sequencing on the PCR product subjected to the quantitative homogenization treatment in the step (S4), constructing a DNA sequence library, and then generating a DNA sequence library report.
Example 4
An eDNA sampling experiment analysis method for a water environment specifically comprises the following steps:
s1, selecting an eDNA filter compatible with any suction pump;
s2, standardization treatment: collecting 97L of water sample from a sampling place, filtering the sampled water by using the eDNA sampler in the step S1, independently packaging the filter, pre-installing a filter membrane, standardizing the filtration parameters on the eDNA samplers of all the samples, and storing after the standardization treatment is finished, wherein the filter membrane of the eDNA filter is made of biodegradable hydrophilic materials, can automatically store the captured eDNA by drying, does not need a filter membrane transfer step, does not need chemical or refrigeration, and is made of polyether sulfone, the thickness of the filter membrane is 1.15 mu m, the diameter of the filter membrane is 48mm, and the filtration parameters on the eDNA samplers of all the samples are standardized at the flow rate of 1.25L/min, the pressure threshold of 11psi and the target volume of 0.6L;
s3, extracting environmental DNA: transferring the experimental sample subjected to the standardization treatment in the step S2 to DNA extraction equipment, adding lytic enzyme, mixing and reacting for 2.7h, and performing centrifugal separation treatment through a centrifugal mechanism to obtain target DNA required by the experiment, wherein the lytic enzyme comprises DNA helicase, DNA gyrase, DNA primer enzyme and DNA synthetase;
s4, PCR amplification homogenization treatment: synthesizing a primer joint by genetic engineering design according to the target DNA sequence obtained in the step S3, carrying out PCR amplification treatment on the designed primer joint through PCR amplification equipment, purifying PCR amplification products, and then carrying out quantitative homogenization treatment on the purified PCR products, wherein all samples need to be treated by an OneStepTMPCR inhibitor removal kit in the PCR amplification process, so that the PCR amplification inhibition effect in the samples is eliminated:
s5, sequencing: and (5) carrying out DNA sequence sequencing on the PCR product subjected to the quantitative homogenization treatment in the step (S4), constructing a DNA sequence library, and then generating a DNA sequence library report.
In conclusion, the invention can realize that the collected experimental sample can be well preserved aseptically by adopting the filtering membrane supported by the biodegradable hydrophilic material and matching with good standardized treatment, thereby well achieving the purpose of rapidly and safely preserving the collected experimental sample, avoiding the occurrence of serious pollution of the collected experimental sample due to nonstandard sampling and preservation in the process of sampling, experimental and analysis of the water environment eDNA, ensuring that the experimental and analysis results are more accurate, and greatly facilitating the analysis work of experimenters.
And those not described in detail in this specification are well within the skill of those in the art.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A method for analyzing an eDNA sampling experiment in a water environment is characterized by comprising the following steps: the method specifically comprises the following steps:
s1, selecting an eDNA filter compatible with any suction pump;
s2, standardization treatment: collecting 90-100L of water samples from a sampling place, filtering the sampled water by using the eDNA sampler in the step S1, independently packaging the filter and pre-installing the filter membrane, standardizing the filtration parameters of the eDNA samplers of all samples, and storing after the standardization treatment is finished;
s3, extracting environmental DNA: transferring the standardized experimental sample obtained in the step S2 to DNA extraction equipment, adding a lytic enzyme, carrying out mixed reaction for 2-3h, and carrying out centrifugal separation treatment through a centrifugal mechanism to obtain the target DNA required by the experiment;
s4, PCR amplification homogenization treatment: synthesizing a primer joint by genetic engineering design according to the target DNA sequence obtained in the step S3, carrying out PCR amplification treatment on the designed primer joint by PCR amplification equipment, purifying PCR amplification products, and then carrying out quantitative homogenization treatment on the purified PCR products:
s5, sequencing: and (5) carrying out DNA sequence sequencing on the PCR product subjected to the quantitative homogenization treatment in the step (S4), constructing a DNA sequence library, and then generating a DNA sequence library report.
2. The method for the experimental analysis of the eDNA sampling for the water environment according to claim 1, characterized in that: the filter membrane of the eDNA filter in step S2 is composed of a biodegradable hydrophilic material, which can automatically preserve the captured eDNA by drying, without a filter membrane transfer step, and without chemical or refrigeration.
3. The method for the experimental analysis of the eDNA sampling for the water environment according to claim 1, characterized in that: in the step S4, all samples need to be subjected to OneStep in the PCR amplification process TM And treating the PCR inhibitor removal kit to eliminate the PCR amplification inhibition effect in the sample.
4. The method for the experimental analysis of the eDNA sampling of the aquatic environment according to claim 2, characterized in that: the filtering membrane is composed of one or more of polyether sulfone, polylactic acid, polyhydroxybutyrate, polyvinyl alcohol, polycaprolactone or poly butylene succinate.
5. The method for the experimental analysis of the eDNA sampling of the aquatic environment according to claim 1, characterized in that: the thickness of the filtering membrane in the step S2 is 1-1.2 mu m, and the diameter is 45-50mm.
6. The method for the experimental analysis of the eDNA sampling of the aquatic environment according to claim 1, characterized in that: in step S2, the filtration parameters on the eDNA samplers for all samples are normalized at a flow rate of 0.5-1.5L/min, a pressure threshold of 8-12psi, and a target volume of 0.3-0.7L.
7. The method for the experimental analysis of the eDNA sampling for the water environment according to claim 1, characterized in that: the lytic enzymes in the step S3 comprise DNA helicase, DNA gyrase, DNA primase and DNA synthetase.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116536433A (en) * | 2023-06-19 | 2023-08-04 | 贵州省生物研究所 | Turbid lake fish monitoring method based on eDNA |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116536433A (en) * | 2023-06-19 | 2023-08-04 | 贵州省生物研究所 | Turbid lake fish monitoring method based on eDNA |
| CN116536433B (en) * | 2023-06-19 | 2023-10-10 | 贵州省生物研究所 | Turbid lake fish monitoring method based on eDNA |
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