CN113528503A - Fixing method for researching structural diversity of freshwater ultramicro eukaryotic algae community - Google Patents
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Abstract
The invention relates to a fixing method for researching community structure diversity of fresh water ultramicro eukaryotic algae, belonging to the technical field of monitoring and researching the cell number and community structure of ultramicro eukaryotic algae in environmental science. Dividing the water into an upper sampling area, a middle sampling area and a lower sampling area according to areas, dividing each area into upper, middle and lower water samples, equally dividing the areas and setting sampling points, sampling every two hours at different sampling points through a water sampler, mixing all the collected water samples to obtain a water sample to be fixed, placing the water sample into a centrifugal tube, adding poloxamer solution as a fixing agent, uniformly mixing and standing to obtain a mixed sample. And placing the mixed sample in liquid nitrogen for quick freezing. And taking out the quick-frozen mixed sample, and putting the quick-frozen mixed sample into an ultra-low temperature refrigerator for refrigeration to obtain a preserved sample. Taking out the preserved sample, and unfreezing to obtain a fixed sample. The invention not only can keep the abundance of the ultramicro eukaryotic algae consistent, but also can keep the diversity and the stable community structure of the ultramicro eukaryotic community, and is suitable for various lake samples.
Description
Technical Field
The invention relates to a fixing method for researching community structure diversity of fresh water ultramicro eukaryotic algae, belonging to the technical field of monitoring and researching the cell number and community structure of ultramicro eukaryotic algae in environmental science.
Background
Due to tiny cells, many groups of super microalgae lack obvious morphological characteristics, and are difficult to distinguish and identify by using a traditional microscopic observation method; the rapid development of molecular biology has not led people to further understand the diversity of ultramicro algae until the beginning of the 21 st century. Depending on the cell structure, microalgae can be classified into prokaryotic microalgae and eukaryotic microalgae. The ultramicro eukaryotic algae is not a certain classification group on the phylogenetic order, but represents a general term of all eukaryotic planktonic algae with the particle size of less than 3 μm, so the species composition of the ultramicro eukaryotic algae is very complex and has extremely high diversity, the ultramicro eukaryotic algae can be the most abundant eukaryotic organisms on the earth, the ultramicro eukaryotic algae in fresh water is not widely concerned until the last few years, and the knowledge on the ultramicro eukaryotic algae is very limited. rRNA gene high-throughput sequencing by flow cytometry combined with 18S is the best method for studying the diversity of ultramicro eukaryotic algae. The measurement is usually performed immediately after the collection of the microalgae sample, and generally, the analysis should be performed immediately after the collection of the sample in order to prevent the cell fluorescence signal from disappearing or autolysis. Because the temperature changes frequently under the room temperature condition, and the cells still continue to carry out cell metabolism in the environment, the metabolic products are accumulated and even acidosis, metabolic disorder and death are caused, and the cells are further decomposed by microorganisms, and finally the result deviation is large. Therefore, the sample should be collected and measured immediately, if the sample cannot be immediately treated, the cell metabolism should be fixed and stopped or inhibited immediately, so that the sample is ensured to be in the initial collection state to the greatest extent, and the measurement error is reduced. The preservation of ultramicro eukaryotic algae samples requires to keep the autofluorescence of the algae on one hand and to ensure the integrity of the cells of the algae at low temperature on the other hand.
Many researches on fixing methods of microalgae and other floating algae are conducted at home and abroad, and currently, common fixing agents for floating algae include Lugol reagent, formalin (containing methanol), ethanol, glutaraldehyde, formaldehyde and the like, but different fixing methods have different influences on algae fluorescence and DNA. Most of the common reagents have great influence on the fluorescence signal or cell morphology of the ultramicro algae cells, such as Lugol reagent, formalin (containing methanol), ethanol and the like, so that the reagents cannot be used for fixing the ultramicro algae samples. Many researchers found that aldehyde solutions, such as formaldehyde, glutaraldehyde, etc., can largely maintain the morphological structure of cells and the integrity of organelles, but still have an influence on the fluorescence signal of algal cells. In particular, only the influence of the fixing methods on the fluorescence characteristics of the ultramicro algae is generally studied, and the influence of sample fixing on the structure of the ultramicro eukaryotic algae community, namely the difference from a fresh sample, is not further explored. Until now, a sample fixing method capable of maintaining the initial community structural characteristics of the ultramicro eukaryotic algae is lacked.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the existing problems and defects, the invention aims to provide a fixing method for researching the structural diversity of the freshwater ultramicro eukaryotic algae community, which can keep the abundance of the ultramicro eukaryotic algae consistent, can also keep the diversity and the community structural stability of the ultramicro eukaryotic community and is suitable for various lake samples.
The technical scheme is as follows: in order to realize the purpose, the invention adopts the following technical scheme: a fixing method for researching structural diversity of freshwater ultramicro eukaryotic algae community comprises the following steps:
step 1: dividing a water body into an upper sampling area, a middle sampling area and a lower sampling area according to areas, dividing each area into upper, middle and lower water samples, equally dividing the areas and setting sampling points, sampling once every two hours at different sampling points through a water sampler, and mixing all the collected water bodies to obtain water samples to be fixed;
step 2: putting the water sample to be fixed obtained in the step 1 into a centrifugal tube, adding poloxamer solution with the volume ratio of 0.01-0.05% as a fixing agent, uniformly mixing and standing to obtain a mixed sample;
and step 3: placing the mixed sample obtained in the step 2 in liquid nitrogen for quick freezing;
and 4, step 4: taking out the quick-frozen mixed sample, and putting the quick-frozen mixed sample into an ultra-low temperature refrigerator for refrigeration to obtain a preserved sample;
and 5: taking out the preserved sample, and unfreezing to obtain a fixed sample.
Further, the poloxamer added in the step 2 accounts for 0.01 percent of the volume.
Further, the liquid nitrogen quick-freezing temperature in the step 3 is-200 to-100 ℃, and the refrigerating temperature of the ultra-low temperature refrigerator in the step 4 is-90 to-70 ℃.
Further, the liquid nitrogen quick-freezing temperature in the step 3 is-196 ℃, and the refrigerating temperature of the ultra-low temperature refrigerator in the step 4 is-80 ℃.
Further, after extracting DNA from the fixed sample obtained in step 5, concentrating the sample by using a magnetic bead method, wherein the magnetic bead method specifically comprises the steps of: adding the magnetic bead solution and the magnetic beads into the thawed preserved sample to obtain a mixed solution, incubating, binding DNA to the magnetic beads, removing supernatant after the mixed solution is clarified, adding nuclease-free water, standing, and concentrating to obtain concentrated DNA.
Further, in the magnetic bead method, magnetic bead liquid is taken out before use, the magnetic bead liquid is kept at room temperature, the magnetic bead liquid is added into a thawed preserved sample after being fully mixed, a pipettor is used for sucking and mixing the magnetic bead liquid uniformly, the preserved sample is placed on a magnetic frame, DNA in the preserved sample is combined onto the magnetic bead through room-temperature incubation, after supernatant liquid is removed, 100-300 mu l of freshly prepared 80% ethanol is added to rinse the magnetic bead, then room-temperature incubation is carried out, the supernatant liquid is removed, the preserved sample is still placed on the magnetic frame, the rinsing step is repeated for 1-2 times, drying is carried out at room temperature for 5-10 min, the treated preserved sample is taken down, nuclease-free water is added, and after uniform mixing, the treated preserved sample is kept still and supernatant liquid is sucked.
Has the advantages that: compared with the prior art, the invention has the following advantages: the method is a novel fixing method for researching the structural diversity of the freshwater ultramicro eukaryotic algae community, has strong adaptability on the premise of maintaining the diversity and the structural stability of the ultramicro eukaryotic community, is suitable for various lake samples, and has good development and utilization prospects.
Drawings
FIG. 1 is a distribution image of ultramicro algae of the present invention in a flow cytometer scattergram;
FIG. 2 is a graph showing the abundance change of ultramicro eukaryotic algae under different immobilization methods of the present invention,
in the figure, (a) is basalt lake, (b) is nidus lake, (c) is small pond, and (d) is pure culture Machilus; FA is formaldehyde, GA is glutaraldehyde, DMSO is dimethyl sulfoxide, and F68 is poloxamer;
FIG. 3 is a Shannon variation diagram of ultramicro eukaryotic algae under different immobilization methods of the invention,
in the figure, (a) is a basalt lake, (b) is a nidus lake, and (c) is a small pond; FA is formaldehyde, GA is glutaraldehyde, DMSO is dimethyl sulfoxide, and F68 is poloxamer;
FIG. 4 Bray-Curtis distance between ultramicro eukaryotic algae colony and initial colony under different immobilization methods of the invention,
in the figure, (a) is a basalt lake, (b) is a nidus lake, and (c) is a small pond; FA is formaldehyde, GA is glutaraldehyde, DMSO is dimethyl sulfoxide, and F68 is poloxamer;
FIG. 5 is a point diagram of the NMDS of ultramicro eukaryotic algae population according to the present invention,
in the figure, (a) is a basalt lake, (b) is a nested lake, and (c) is a small pond point shape representing a fixed time, and a point color representing a fixed method.
Detailed Description
The present invention is further illustrated by the following figures and specific examples, which are to be understood as illustrative only and not as limiting the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalent modifications thereof which may occur to those skilled in the art upon reading the present specification.
Examples
(1) Selecting water samples of the great lakes, the basalt lakes and the small landscape ponds as field samples for testing, and selecting pure McKe algae (algae)Mychonastes homosphaera) As a control for ultramicro eukaryotic algae;
(2) collecting water bodies in all water areas by using an organic glass water sampler, uniformly mixing water samples and bottling;
(3) adding poloxamer accounting for 0.01 percent of the volume into a water sample, placing the water sample into a 15mL sterile centrifuge tube, uniformly mixing, standing for 15min in a dark place at room temperature, quickly freezing the sample by liquid nitrogen, and freezing and storing the sample at-80 ℃.
(4) Setting fixed time: fresh water samples are used as a control, and 3 preservation times of 30 days, 90 days and 180 days are set.
(5) Sorting samples by a flow cytometer: and naturally dissolving the quick-frozen sample at room temperature. The set-up conditions for the flow cytometer were as follows: according to the autofluding fluorescence difference of the super microalgae, selecting fluorescence FL3 and FL5 channels (excitation wavelengths of 488 nm and 640 nm) chlorophyll fluorescence; the side scattered light (FSC) was set according to the size of the ultramicro algal cells, and a measurement region within 3 μm was defined using a 3.1 μm niele (SPHEROTM URFP-30-2, Spherotech, Calif., USA) bead solution. After being uniformly mixed, the sample to be detected is firstly prefiltered by a 300-mesh bolting silk, and then is injected with low-speed 200 plus 300 cells.
According to the defined range, the flow cytometer image results of the microalgae groups are obtained as shown in fig. 1, the PPEs and PCY cells are sorted into an Eppendorf centrifuge tube by adopting an Enrichment mode (Enrichment Model), samples in each time period are sorted to the same number according to the concentration counted by the flow cytometer, and the samples are stored at-20 ℃ for subsequent DNA extraction.
(6) Sorted sample DNA extraction, PCR and high throughput sequencing
Sample DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen).
The DNA concentration of the flow-sorting algae cells is low, the DNA needs to be concentrated by a magnetic bead method, the magnetic bead solution is taken out from 2-8 ℃ in advance for 30 min, and the magnetic bead solution is kept stand to enable the temperature to be balanced to the room temperature. And (3) reversing or vortex oscillating to fully and uniformly mix the magnetic bead solution, sucking a certain volume (specifically according to the sample condition, referring to the DNA purification reference condition) of the magnetic bead solution, adding the magnetic bead solution into the DNA sample, and slightly sucking and beating the DNA sample for 10 times by using a pipettor to fully and uniformly mix the magnetic bead solution. The incubation was performed at room temperature for 10min to allow the DNA to bind to the magnetic beads. The sample was placed on a magnetic stand and after the solution cleared (about 5 min), the supernatant was carefully removed. Keeping the sample on the magnetic stand all the time, add 200 μ l of freshly prepared 80% ethanol to rinse the beads, incubate for 30 sec at room temperature, carefully remove the supernatant. Repeat step 5 once for a total of two rinses. Keeping the sample on the magnetic frame all the time, and opening the cover to dry the magnetic beads for about 5-10 min at room temperature. Taking out the sample from the magnetic frame, adding appropriate amount of nuclease-free water, vortex vibrating or blowing with a pipette, mixing well, and standing at room temperature for 2 min. Standing on a magnetic frame for 5min until the solution is clarified, carefully sucking the supernatant into a new nuclease-free centrifuge tube. After concentration, the DNA concentration is increased from 0.6-1.6 ng/muL to 3-10 ng/muL.
The concentration of DNA of the flow-sorting algae cells is low, the magnetic bead method is needed to concentrate the DNA, and the specific operation is carried out according to VAHTS®DNA Clean Beads (Vazyme) instructions.
The eukaryotic universal primer pair EK-NSF573 is adopted: 5'-CGCGGTAATTCCAGCTCCA-3', Ek-NSR 951: 5'-TTGGYRAATGCTTTCGC-3', performing high fidelity PCR amplification on the 18S rRNA V4 region of the detection region for 25 cycles. 3 replicates were set up with standard bacterial/bacterial genomic DNA Mix as positive control. Subsequently, 3 parallel amplification products from the same sample were mixed and purified by adding an equal volume of nucleic acid purification magnetic bead AgencourtAMpure XP (Beckman). After a specific tag sequence (a section of sequence which is introduced for distinguishing samples when a plurality of samples are subjected to mixed sequencing and marks sample source information) is added to each sample, the library is subjected to quantification, mixing and quality detection, and then the library is subjected to sequencing by adopting an Illumina MiSeq high-throughput sequencing platform and a 2 x 250 bp double-end sequencing strategy.
(7) High throughput sequencing data processing and data analysis
The quality control and clustering process of the original sequence obtained by sequencing is as follows: a. removing sequences with the end quality lower than 20, removing adapter sequences and sequences with the length less than 100 by using TrimGalore software; b. carrying out double-end combination by using FLASH2 software to obtain an effective sequence; c. removing primers at two ends of the sequence by using mortur v.1.39.3 software; d. removing sequences with total base error rate more than 2 and length less than 100 bp by using usearch software; e. removing the singleton sequence and the Chimera sequence by using UPARSE software, clustering the sequences into different OTUs (operational Taxonomic units) according to the 97% similarity, and finally annotating the clustered OTUs according to a Silva database.
Performing relevant statistical analysis on the obtained OTUs by using R3.3.2 (http:// cran.r-project.org), and firstly calculating the diversity index of the sample by using the sample after the re-extraction; NMDS analysis of community characteristics is carried out by utilizing vegan bags based on Bray-Curtis distance, and ANOSIM analysis of difference significance is carried out; the response of different groups of microorganisms in the sequenced samples to the immobilization method was explored using R-pack edgeR and pheatmap (P < 0.001). Statistical analysis of the significance of differences in fixed samples of microalgae (e.g., differences in diversity, differences in algal density, and differences in relative abundance)
Results obtained in the examples:
(1) effect of immobilization method on abundance of ultramicro algae
As shown in fig. 2, different immobilization methods and immobilization times have an effect on the abundance of ultramicro eukaryotic algae sorted by flow cytometer. In basalt lake, the abundance of ultramicro eukaryotic algae decreases significantly (P < 0.05) with prolonged fixation time. The abundance of ultramicro eukaryotic algae of the two fixing methods of 10% DMSO and 10% DMSO +0.01% F68 is significantly lower than that of the other methods (P < 0.05), while the abundance of ultramicro eukaryotic algae of the three fixing methods of 2% formaldehyde, 2% formaldehyde +0.01% F68, 0.25% glutaraldehyde +0.01% F68 is significantly higher than that of the other methods (P < 0.05), wherein 2% formaldehyde +0.01% F68 does not significantly decrease after 30 days of fixing (P < 0.05). In the nested lakes, the abundance of ultramicro eukaryotic algae changes in different trends with the fixing time. The abundance of the ultramicro eukaryotic algae obtained by the two fixing methods of 10% DMSO and 10% DMSO +0.01% F68 is remarkably reduced (P is less than 0.05), and the abundance of the ultramicro eukaryotic algae is extremely low when the ultramicro eukaryotic algae is fixed for 30 days. In the other four fixing methods, the ultramicro eukaryotic algae are remarkably increased, and the abundance difference among the ultramicro eukaryotic algae is not remarkable (P is more than 0.05). In a small pond, the abundance of the ultramicro eukaryotic algae of the two fixing methods of 10% DMSO and 10% DMSO +0.01% F68 is still reduced rapidly (P is less than 0.05), and except that the abundance of the ultramicro eukaryotic algae of the 0.01% F68 is increased remarkably after 180 days of fixing, the abundance of the ultramicro eukaryotic algae is not changed remarkably along with the increase of the fixing time of the other fixing methods. For the genus Macaca in a pure culture system, the algae abundance of the two fixing methods of 10% DMSO and 10% DMSO +0.01% F68 is remarkably reduced (P is less than 0.05), and especially the abundance of the genus Macaca is extremely low when the fixing is carried out for 90 days. While the marcescens' abundances of the two fixation methods of 2% formaldehyde and 0.01% F68 tended to increase and then decrease, the three fixation methods of 0.25% glutaraldehyde, 2% formaldehyde +0.01% F68 and 0.25% glutaraldehyde +0.01% F68 decreased slightly but did not differ significantly from the initial algae abundance (P > 0.05) with the duration of the fixation.
(2) Influence of fixing method on ultramicro eukaryotic algae community structure
We extracted DNA from samples treated by different immobilization methods and performed high-throughput sequencing, and except that 10% DMSO and 10% DMSO +0.01% F68 are used as impurity signals to interfere seriously, so that the flow cytometer can not sort the super-microalgae cells, and the sequencing of other samples is successful.
As shown in FIG. 3, different immobilization methods and immobilization times have an effect on the α diversity of ultramicro eukaryotic algae. In basalt lakes, the ultramicro eukaryotic algae α diversity of 2% formaldehyde and 2% formaldehyde +0.01% F68 did not differ significantly with increasing fixation time (P > 0.05). The alpha diversity of the ultramicro eukaryotic algae of 0.25% glutaraldehyde and 0.25% glutaraldehyde +0.01% F68 fluctuated up and down compared with the initial sample, while the alpha diversity of the ultramicro eukaryotic algae of 0.01% F68 was significantly higher than the initial sample. For the nido lake sample, the ultramicro eukaryotic algae alpha diversity of various immobilization methods is significantly higher than that of the original sample. Among them, the α diversity of ultramicro eukaryotic algae of the 2% formaldehyde group was significantly increased at the time of fixation for 30 days, and then did not significantly change with the increase of the fixation time (P > 0.05). The alpha diversity of ultramicro eukaryotic algae was still not significantly changed (P > 0.05) by 0.25% glutaraldehyde +0.01% F68 and 0.01% F68 at 30 days of fixation, but it was significantly changed with time (P < 0.05). For small ponds, the alpha diversity of the various fixation methods decreased significantly (P < 0.05) at 30 days and then increased significantly (P < 0.05). The alpha diversity of 0.01% F68 decreased significantly less than the other methods (P < 0.05).
The Bray-Curtis distance is used to compare the differences in composition of two populations of microorganisms based primarily on the statistical counts of OTUs, and as shown in FIG. 4, the Bray-Curtis distance is used to measure the differences in composition of ultramicro eukaryotic algae populations of different immobilization methods from the initial ultramicro eukaryotic algae composition. In basalt lakes, the Bray-Curtis distance of 0.01% F68 was significantly less than other fixation methods, except for the Bray-Curtis distance of 0.25% glutaraldehyde +0.01% F68 at 30 days of fixation. The Bray-Curtis distance for this fixation method of 0.01% F68 was also significantly less than for other fixation methods in the brood lake and lagoon.
As shown in FIG. 5, the tendency of the population of ultramicro eukaryotic algae to change under different immobilization methods was compared at the OTU level using the NMDS analysis based on the Bray-Curtis distance. In each lake zone sample, the ultramicro eukaryotic algae community of the 0.01% F68 group is similar to the initial community point and the community structure is similar. In addition, except for the samples fixed for 30 days in the basalt lake by adopting 0.25% of glutaraldehyde and 0.01% of F68, the colony structures of the other lake area samples are far away from the initial colony structure, and the structural difference is obvious. Except for basalt lake, samples fixed at 0.01% F68 were divided into three structural groups of 30 days, 90 days and 180 days, respectively, according to the fixed time. To verify the accuracy of this class group partitioning, we performed verification using ANOSIM analysis. There was no significant difference in the structure of fresh samples and 0.01% F68 immobilized ultramicro eukaryotic algae colonies in each lake (basalt lake: P = 0.138; nido lake: P = 0.369; small pond: P = 0.407), which were significantly different from the colony structures of other immobilization methods (basalt lake: P < 0.001; nido lake: P < 0.001; small pond: P < 0.001). In basalt lake, the change of ultramicro eukaryotic algae community structure with fixed time is not obvious (P is more than 0.05). In the nido lake and the small pond, the change of the structure of the ultramicro eukaryotic algae community of the sample which is not fixed by 0.01 percent F68 along with the fixed time is obvious (P < 0.05).
Therefore, the community structure change of the ultramicro eukaryotic algae is different after the ultramicro eukaryotic algae is fixed by different methods. Compared with other fixing methods, the 0.01 percent F68 can keep the abundance of the ultramicro eukaryotic algae consistent, can also keep the diversity and the community structure stability of the ultramicro eukaryotic community, and is suitable for various lake samples. Therefore, fixation using 0.01% F68 is the most effective fixation method in research on diversity of ultramicro eukaryotic algae.
Claims (6)
1. A fixing method for researching the structural diversity of freshwater ultramicro eukaryotic algae community is characterized in that: the method comprises the following steps:
step 1: dividing a water body into an upper sampling area, a middle sampling area and a lower sampling area according to areas, dividing each area into upper, middle and lower water samples, equally dividing the areas and setting sampling points, sampling once every two hours at different sampling points through a water sampler, and mixing all the collected water bodies to obtain water samples to be fixed;
step 2: putting the water sample to be fixed obtained in the step 1 into a centrifugal tube, adding poloxamer solution with the volume ratio of 0.01-0.05% as a fixing agent, uniformly mixing and standing to obtain a mixed sample;
and step 3: placing the mixed sample obtained in the step 2 in liquid nitrogen for quick freezing;
and 4, step 4: taking out the quick-frozen mixed sample, and putting the quick-frozen mixed sample into an ultra-low temperature refrigerator for refrigeration to obtain a preserved sample;
and 5: taking out the preserved sample, and unfreezing to obtain a fixed sample.
2. The fixed method for researching structural diversity of freshwater ultramicro eukaryotic algae community according to claim 1, characterized in that: the poloxamer added in the step 2 accounts for 0.01 percent of the volume.
3. The fixed method for researching structural diversity of freshwater ultramicro eukaryotic algae community according to claim 1, characterized in that: the liquid nitrogen quick-freezing temperature in the step 3 is-200 to-100 ℃, and the refrigerating temperature of the ultra-low temperature refrigerator in the step 4 is-90 to-70 ℃.
4. The method according to claim 3, wherein the method comprises the following steps: the liquid nitrogen quick-freezing temperature in the step 3 is-196 ℃, and the refrigerating temperature of the ultra-low temperature refrigerator in the step 4 is-80 ℃.
5. The fixed method for researching structural diversity of freshwater ultramicro eukaryotic algae community according to claim 1, characterized in that: and (5) extracting DNA from the fixed sample obtained in the step (5), and concentrating by using a magnetic bead method, wherein the magnetic bead method comprises the following specific steps: adding the magnetic bead solution and the magnetic beads into the thawed preserved sample to obtain a mixed solution, incubating, binding DNA to the magnetic beads, removing supernatant after the mixed solution is clarified, adding nuclease-free water, standing, and concentrating to obtain concentrated DNA.
6. The method of claim 5, wherein the method comprises the following steps: the magnetic bead liquid in the magnetic bead method is taken out before use, the magnetic bead liquid is kept stand to room temperature, the magnetic bead liquid is added into a thawed preserved sample after being fully and uniformly mixed, a pipettor is used for sucking and mixing the magnetic bead liquid uniformly, the preserved sample is placed on a magnetic frame, DNA in the preserved sample is combined onto the magnetic bead through room-temperature incubation, after supernatant liquid is removed, 100-300 mu l of freshly prepared 80% ethanol is added to rinse the magnetic bead, then room-temperature incubation is carried out, the supernatant liquid is removed, the preserved sample is still placed on the magnetic frame, the rinsing step is repeated for 1-2 times, drying is carried out for 5-10 min at room temperature, the treated preserved sample is taken down, nuclease-free water is added, and standing and supernatant liquid is absorbed after uniform mixing is carried out.
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| CN113528503B (en) | 2023-03-24 |
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