Environmental DNA species monitoring method based on Chinese sturgeons

Abstract

The invention discloses an environmental DNA species monitoring method based on Chinese sturgeons, and relates to the technical field of biological gene detection.According to the environmental DNA species monitoring method based on the Chinese sturgeons, mitochondrial whole genomes of the Chinese sturgeons are determined through a method combining a second-generation library-building sequencing technology and a third-generation library-building sequencing technology, and the whole genomes of the mitochondria of the Chinese sturgeons are determined through probe design and PCR amplification sequencing. The sequence of the whole mitochondrial genome of the Chinese sturgeons is combined for comparison, and the specificity difference between the Chinese sturgeons and other related sturgeons is distinguished, so that the efficiency of detecting rare and endangered species Chinese sturgeons by an environmental DNA method is improved, and the accuracy of species identification is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological gene detection, and particularly relates to an environmental DNA species monitoring method based on Chinese sturgeon. BACKGROUND

[0002] The existing environmental DNA species monitoring method based on Chinese sturgeon still has the following problems in actual use:

[0003] Due to the objective reality that the number of rare and endangered species individuals is extremely small and the appearance frequency and activity frequency are low, in the process of global biodiversity protection projects, most of them rely on species identification methods based on traditional morphology in the set quadrat, and the traditional identification method is mainly based on the morphological characteristics of the appearance of the organism, which is a physical means-based judgment method. This method is largely dependent on the experience of the identification personnel, especially when identifying early resources of some aquatic organisms, the traditional method will encounter some bottlenecks.

[0004] As a national first-class wild aquatic protected animal, Chinese sturgeon is a large-scale river-homing spawning fish born in the Yangtze River, fattened in the sea, and finally returned to the Yangtze River for reproduction. It is a flagship species and umbrella species in the Yangtze River aquatic ecosystem. However, in recent decades, with the intensification of human activities, global climate change and species evolution factors, the Chinese sturgeon population has been assessed as critically endangered by the World Conservation Union. Based on the above basic situation, it is particularly important to monitor the Chinese sturgeon breeding population in the only spawning ground and protection zone downstream of Gezhouba Dam, other main stream sections of the Yangtze River and the first-order tributary Qingjiang River section of the Yangtze River. SUMMARY

[0005] The present application aims to provide an environmental DNA species monitoring method based on Chinese sturgeon to solve the above problems.

[0006] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme: an environmental DNA species monitoring method based on Chinese sturgeon, comprising the following steps:

[0007] S1, weighing 500mg of fresh tail fin tissue or frozen blood sample of Chinese sturgeon;

[0008] S2, placing the sample in SDS lysis solution, and adding appropriate amount of proteinase K and mercaptoethanol, and mixing gently to promote lysis;

[0009] S3, after lysis, cooling the sample to room temperature, and then centrifuging;

[0010] S4, after centrifugation, adding chloroform / isopentanol to extract twice;

[0011] S5, precipitate the DNA using isopropanol, mix gently and invert, then centrifuge again;

[0012] S6, discard the waste liquid, and wash the DNA precipitate twice with 75% ethanol;

[0013] S7, after the DNA precipitate is dried, 200 μl of EB solution is added to dissolve it, and RNAse digestion is performed;

[0014] S8, purify the DNA using an OMEGA purification column;

[0015] S9, after purification, further purification is performed using Ampure XP beads;

[0016] S10, the quality of the extracted DNA is detected by Nanodrop, Qubit, and electrophoresis methods;

[0017] S11, the mitochondrial whole genome of Acipenser sinensis is determined using a combination of second-generation library sequencing technology and third-generation library sequencing technology, and is compared in parallel with the mitochondrial genome of Acipenser sinensis uploaded to NCBI;

[0018] S12, by probe design and PCR amplification sequencing, the target DNA sequence of Acipenser sinensis is specifically recognized and combined, and Acipenser sinensis is distinguished from other related species, which is used for subsequent verification analysis experiments.

[0019] Further, the second-generation library sequencing technology comprises the following steps:

[0020] After the DNA sample is detected and confirmed to be qualified, a Covaris ultrasonic crusher is used for random fragmentation treatment;

[0021] After a series of steps of end repair, A tail addition, sequencing adapter ligation, purification, and PCR amplification, the construction of the library is completed;

[0022] After the library construction is completed, the Qubit 3.0 is first used to determine the concentration, and the library is appropriately diluted;

[0023] The Agilent 2100 instrument is used to detect the insert size of the library, to ensure that the fragment size meets the expected requirements;

[0024] The BioRAD CFX 96 fluorescence quantitative PCR instrument is used, together with the Bio-RAD KIT iQ SYBR GRN reagent, to perform QPCR analysis, to accurately determine the effective concentration of the library, thereby ensuring the quality of the sequencing library;

[0025] After the library is confirmed to be qualified through detection, different libraries are mixed according to effective concentrations and target data volume requirements;

[0026] The clustering process is performed on the flowcell by cBOT, and a double-end sequencing program is performed on the NovaSeq 6000 sequencing platform to obtain sequence reads with a length of 150bp.

[0027] Further, the third-generation library construction sequencing technology comprises the following steps:

[0028] 2.5ug of the qualified DNA sample is selected, 1x magnetic beads are used for purification treatment, and then 1ul of the sample is taken for quantitative analysis by Qubit;

[0029] The DNA is subjected to damage repair and end processing, and the incubation conditions are 20 DEG C for 10 minutes, 65 DEG C for 10 minutes, and 4 DEG C storage;

[0030] The DNA is purified again using 1x magnetic beads, and eluted with 61ul of EB buffer, and then 1ul of the sample is taken for Qubit quantification;

[0031] The sequencing adapter is connected to the DNA sample, and the incubation conditions are 25 DEG C for 10 minutes;

[0032] The DNA is purified using 0.4x magnetic beads, eluted using 25ul of Elution buffer, and then 1ul of the sample is taken for Qubit quantification;

[0033] The sequencing library is prepared for machine use;

[0034] The prepared library is loaded into the R9.4 sequencing chip, and the PromethION sequencer is used for sequencing for 48-72 hours.

[0035] Compared with the prior art, the environmental DNA species monitoring method based on Acipenser sinensis provided by the application has the following beneficial effects:

[0036] The environmental DNA species monitoring method based on Acipenser sinensis combines the second-generation library construction sequencing technology and the third-generation library construction sequencing technology to determine the mitochondrial whole genome of Acipenser sinensis, and compares the sequences of the whole mitochondrial genome of Acipenser sinensis with the sequences of other related sturgeons to distinguish the specific differences between Acipenser sinensis and other related sturgeons, thereby improving the efficiency of the environmental DNA method in detecting the rare and endangered species Acipenser sinensis and improving the accuracy of species identification. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to make the technical scheme of the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application. Those skilled in the art can also obtain other accompanying drawings according to these accompanying drawings.

[0038] Figure 1 A Chinese sturgeon mitochondrial genome map is completed for the present application;

[0039] Figure 2 A one-dimensional droplet distribution map is for the present application;

[0040] Figure 3 A one-dimensional scatter plot of the detection results of a certain microdroplet digital PCR of a sample with different concentrations is for the present application;

[0041] Figure 4 A Chinese sturgeon DNA nucleic acid standard sample fitting map under different concentrations is for the present application;

[0042] Figure 5 A Chinese sturgeon and other sturgeon sequence alignment map is for the present application;

[0043] Figure 6 A reverse probe sequence scoring system page map is for the present application. DETAILED DESCRIPTION

[0044] In order for those skilled in the art to better understand the technical scheme of the present application, the present application will be further described in detail below with reference to the accompanying drawings.

[0045] Please refer to Figure 1-6 A Chinese sturgeon-based environmental DNA species monitoring method, comprising the following steps:

[0046] S1, weigh 500 mg of fresh tail fin tissue or frozen blood sample of Chinese sturgeon;

[0047] S2, place the sample in SDS lysis solution, and add appropriate amount of proteinase K and mercaptoethanol, and mix gently to promote lysis;

[0048] S3, after lysis is completed, cool the sample to room temperature, and then perform centrifugal treatment;

[0049] S4, after centrifugation, take the supernatant, and add chloroform / isopentanol (ratio 24:1) for twice extraction;

[0050] S5, use isopropyl alcohol to precipitate DNA, mix gently after mixing, and then perform centrifugation again;

[0051] S6, discard the waste liquid, and wash the DNA precipitate with 75% ethanol twice;

[0052] S7, after the DNA precipitation is aired dry, 200ul EB solution is added for dissolution, and RNAase digestion treatment is carried out;

[0053] S8, the DNA is purified by using an OMEGA purification column;

[0054] S9, after the purification is completed, Ampure XP beads are further used for purification;

[0055] S10, the extracted DNA is detected in quality by a Nanodrop, Qubit and electrophoresis method;

[0056] S11, the mitochondrial whole genome of Acipenser sinensis is determined by combining the second-generation library sequencing technology and the third-generation library sequencing technology, and is compared in parallel with the mitochondrial genome of Acipenser sinensis uploaded by NCBI;

[0057] S12, by probe design and PCR amplification sequencing, the target DNA sequence of Acipenser sinensis is specifically recognized and combined, Acipenser sinensis is distinguished from other related species, and is used for subsequent verification analysis experiment.

[0058] The second-generation library sequencing technology comprises the following steps:

[0059] After the DNA sample is detected and confirmed to be qualified, a Covaris ultrasonic wave crusher is used for random fragmentation treatment;

[0060] After a series of steps such as end repair, A tail addition, connection of sequencing adapters, purification and PCR amplification, the construction of the library is completed;

[0061] After the library construction is completed, the Qubit 3.0 is first used for preliminary concentration determination, and the library is appropriately diluted;

[0062] The Agilent 2100 instrument is used to detect the insert size of the library, and it is ensured that the fragment size meets the expected requirements;

[0063] The BioRAD CFX 96 fluorescence quantitative PCR instrument is used, and the Bio-RAD KIT iQ SYBR GRN reagent is used for QPCR analysis, so as to accurately determine the effective concentration of the library (ensure that the effective concentration of the library is more than 10nM), thereby ensuring the quality of the sequencing library;

[0064] After the library is detected and confirmed to be qualified, different libraries are mixed according to the effective concentration and target data volume requirement;

[0065] The clusterization process was performed on a flowcell by cBOT and a double-end sequencing procedure (PE150) was performed on a NovaSeq 6000 sequencing platform to obtain sequence reads of 150 bp in length.

[0066] The third-generation library preparation sequencing technology comprises the following steps:

[0067] 2.5 μg of the qualified DNA sample was selected and purified by 1x magnetic beads, and then 1 μl of the sample was taken for quantitative analysis by Qubit;

[0068] The DNA was subjected to damage repair and end processing,

[0069] DNA 48ul NEBNext FFPE buffer (NEB) 3.5ul NEBNext End-prep buffer (NEB) 2ul FFPE DNA Repair Mix (NEB) 2ul End-prep enzyme Mix (NEB) 3.5ul Total 60ul

[0070] Acipenser sinensis and other related acipenseriformes sequences

[0071] Incubation conditions: 20°C for 10 min, 65°C for 10 min, and 4°C storage;

[0072] The DNA was purified again using 1x magnetic beads and eluted with 61 μl of EB buffer, and then 1 μl of the sample was taken for Qubit quantification;

[0073] The sequencing adapter was connected to the DNA sample,

[0074] Pooling DNA 60ul Ligation buffer (SQK-LSK110) 25ul Adapter Mix (SQK-LSK110) 5ul Quick T4 DNA ligase (NEB) 10ul Total 100ul

[0075] Acipenser sinensis and other related acipenseriformes sequences

[0076] Incubation conditions: 25°C for 10 min;

[0077] The DNA was purified using 0.4x magnetic beads and eluted using 25 μl of Elution buffer (model SQK-LSK110), and then 1 μl of the sample was taken for Qubit quantification;

[0078] The sequencing library was prepared for machine use;

[0079] DNA with added sequencing adapter 24ul LB (SQK-LSK110) 51ul SQB (SQK-LSK110) 75ul Total 150ul

[0080] Acipenser sinensis and other related acipenseriformes sequences

[0081] The prepared library was loaded into an R9.4 sequencing chip and sequenced for 48-72 hours using a PromethION sequencer (Oxford Nanopore Technologies, Oxford, UK).

[0082] Basic bioinformatics analysis:

[0083] 1)Mitochondrial genome complete sequencing data statistics: combined with Illumina Hiseq and three generation ONT sequencing technology, the complete sequencing of mitochondrial genome was successfully completed; the sequencing library suitable for Illumina and ONT was constructed, and the obtained sequencing data was strictly controlled; and the bioinformatics analysis means was used to draw the complete map of mitochondrial genome.

[0084] 2)Sequencing data quality control process: the original sequencing data often contains sequencing adapter sequences, low-quality reads, high-N rate sequences and too short sequences, which will affect the quality of subsequent analysis; in order to ensure the accuracy of bioinformatics analysis, the original sequencing data was filtered to obtain high-quality sequencing data (clean data);

[0085] The specific steps are as follows:

[0086] (a) First, remove the adapter sequences in reads, and the reads without insert fragments caused by adapter self-connection; (b) then, quality trimming is performed on the sequence end (3' end), and the bases with quality value less than 20 are removed; if there are still bases with quality value less than 10 in the trimmed sequence, the whole sequence is rejected, otherwise it is retained; (c) then, remove the reads containing N rate more than 10%; (d) finally, discard the sequences with length less than 20 bp after adapter removal and quality trimming.

[0087] 3)Genome assembly: only fastq file is needed, MitoZ can automatically complete the analysis, and output annotated mitochondrial gene (Genbank format) and chart.

[0088] Software: MitoZ (https: / / gitee.com / CHANyp / MitoZ#citation)

[0089] 4)Gene prediction: use MITOS software to predict mitochondrial genes.

[0090] Software: MITOS (http: / / mitos2.bioinf.uni-leipzig.de / index.py)

[0091] 5)Genome structure map: use MitoZ to visualize the physical map of complete mitochondrial genome sequence.

[0092] Software: MitoZ (https: / / gitee.com / CHANyp / MitoZ#citation)

[0093] Mitochondrial genome of Acipenser sinensis:

[0094] The determination result is that the full-length of the mitochondrial genome sequence of Acipenser sinensis is 16680 bp; the mitochondrial genome of adult Acipenser sinensis is similar to that of most aquatic animals, and the morphological structure is a circular molecule, the arrangement order is basically consistent with that of Acipenser sinensis on NCBI data, the arrangement mode is relatively compact, and it contains 13 protein-coding genes, 22 transfer RNAs (tRNA), 2 ribosomal RNAs (rRNA), 1 light chain replication initiation region (OL), and 1 non-coding control region (D-loop region); through sequence statistical analysis, it is found that the average content of the four bases of the mitochondrial genome of Acipenser sinensis is: the content of A is 30.3%, the content of C is 29.2%, the content of G is 16.4%, and the content of T is 24.1%, and the sequence is similar to the mitochondrial genome of vertebrates, the GC content of the mitochondrial genome sequence of Acipenser sinensis is 45.6%, which is slightly less than the AT base content of 54.4%, indicating that its mitochondrial genome has base AT preference.

[0095] Example 1 DNA verification analysis:

[0096] The fin bar and muscle tissue of adult Acipenser sinensis, Acipenser dabryanus, Acipenser schereri and hybrid sturgeon were taken, and the DNA of the four kinds of sturgeons was extracted. By measuring the sequence of the full mitochondrial genome of Acipenser sinensis, the mitochondrial D-loop sequence of other four kinds of sturgeons was obtained by searching the NCBI nucleic acid redundancy database, and compared with the above-mentioned sequencing mitochondrial genome sequence of Acipenser sinensis. It is found that there are four specific differences in different sturgeons at the 184bp start to the downstream 20bp or so, with Acipenser sinensis as the reference sequence. The analysis results are as follows: the mitochondrial D-loop sequence of the following species was obtained by searching the NCBI nucleic acid redundancy database, and was compared to find that there are four specific differences in different sturgeons at the 184bp start to the downstream 20bp or so, with Acipenser sinensis as the reference sequence. The analysis results are shown in Figure 5

[0097] The specific sequence characteristics of this region are shown in the following table:

[0098] Species Sequence Analysis Acipenser sinensis G******A*C*****A Acipenser dabryanus A******A*T*****G Acipenser schrenckii T******G*C*****G Acipenser baerii x Acipenser schrenckii A******A*T*****G

[0099] Sequence table of Acipenser sinensis and other related sturgeons

[0100] ​According to the above sequence characteristics, the probe designed for Acipenser sinensis is as follows: the red base is LNA modified, 5'-HEX-CTACCTTGTTGTTCTTCTCT-MGB-3' (antisense strand probe). Therefore, according to the above sequence characteristics, the probe and primer are designed for the antisense strand of the reference sequence.

[0101] The product size of the antisense strand probe is 120 bp

[0102] Primer-f: 5'-TCATTCCCTCGAGCAGTTGTG-3'

[0103] Primer-r1: 5'-CATGCCAAGAATCTTGCCAAC-3'

[0104] Probe2: 5'-CTACCTTGTTGTTCTTCTCT-3'

[0105] The antisense strand probe and primer designed by us are scored by the Primer Probe Test Tool module, and the specific score is shown in Table 1. Figure 6

[0106] Verification of probe specificity

[0107] The DNA of the four fin strips of sturgeon mentioned above was extracted, and the sample copy number was determined by microdroplet digital PCR (ddPCR) quantification method; the sample was quantified by SNPER DQ24 digital PCR; the probe used was the antisense strand probe designed above; the dPCR system was 20 μl, and the reagent was 2X dPCR MasterMix (Probe) (Sniper).

[0108] reagents Sample volume 2X dPCR MasterMix (Probe) 12.5 μl Primer-f 1 μl Primer-r 1 μl Probe 0.5 μl DNA 3.0 / 1.5 ng ddH2O to 20 μl

[0109] ddPCR reaction system

[0110] PCR amplification procedure:

[0111] 60℃ constant temperature for 5 min, 98℃ hot start for 15 min, 1 cycle; 95℃ denaturation for 20 s, 60℃ extension for 30 s, 40 cycles; 60℃ constant temperature for 1 min; fluorescence data acquisition was performed at the end of PCR reaction; PCR was performed on a DQ24 digital PCR instrument; finally, the specificity of the probe was determined by counting the digital droplet PCR results of the above four kinds of sturgeon.

[0112] Accuracy verification and determination of detection limit

[0113] ​The limit of quantitation (LOQ) was determined using the prepared Chinese sturgeon standard DNA stock solution. The specific method was as follows: the copy number concentration of the prepared nucleic acid stock solution was determined, and then the original standard nucleic acid stock solution of known concentration was diluted using the equal-fold serial dilution method. The concentration changes were successively 1 / 2, 1 / 4, 1 / 8, 1 / 16... up to the 14th dilution. Then, the diluted Chinese sturgeon standard at the 14 known concentration gradients was analyzed using the ddPCR method.

[0114] Probe specificity verification

[0115] Two parallel ddPCR experiments were conducted using DNA from four different sturgeon species at two different concentrations (as shown in Table 1.1). Figure 2 As shown in Table 1.2, the probe designed in this study was optimized for Chinese sturgeon (Acipenser sinensis) and performed well in ddPCR analysis, exhibiting excellent fluorescence signals. Except for two different concentrations of Chinese sturgeon DNA samples that produced positive droplets in ddPCR experiments, no positive droplets were produced in droplet digital polymerase chain reactions of the other three sturgeon species at two different DNA concentrations. To ensure the rigor of the probe specificity conclusion and to prevent false positives, we recovered the positive product chips from the ddPCR reactions of the aforementioned Chinese sturgeon environmental samples and then sequenced the products, comparing them with the corresponding base sequences of Chinese sturgeon in Genebank on NCBI. The comparison results were consistent. Based on the results of the amplification and sequencing experiments, it is demonstrated that the probe designed in this study can successfully distinguish Chinese sturgeon from three other closely related sturgeon species, further improving the efficiency of environmental DNA methods in detecting the rare and endangered species Chinese sturgeon and enhancing the accuracy of species identification. It can be applied to subsequent ddPCR experiments on field environmental samples. Based on the probe sequence information and probe design requirements, the designed antisense probe was ultimately applied to subsequent quantitative experiments in the field environment.

[0116] Serial Number Sample Name DNA concentration (ng / μl) 1 1A_Chinese Sturgeon 1.5 2 1B_Hybrid Sturgeon 1.5 3 1C_Sturgeon schlegelii 1.5 4 1D_Yangtze Sturgeon 1.5 5 1E_Chinese Sturgeon 3.0 6 1F_Hybrid Sturgeon 3.0 7 1G_Shi Changxun 3.0 8 1H_Sturgeon dell'Hercules 3.0

[0117] Table 1.1 Concentration of Experimental Samples

[0118] Sample Name Total number of droplets Dilution times Concentration (cp / ul) Positive droplet count negative droplet count 1A_Chinese Sturgeon 20225 1 320.32 4572 15653 1B_Hybrid Sturgeon 20389 1 0.00 0 20389 1C_Sturgeon schlegelii 20976 1 0.00 0 20976 1D_Yangtze Sturgeon 20476 1 0.00 0 20476 1E_Chinese Sturgeon 18103 1 645.66 7303 10800 1F_Hybrid Sturgeon 21121 1 0.00 0 21121 1G_Shi Changxun 18886 1 0.00 0 18886 1H_Yangtze Sturgeon 19519 1 0.00 0 19519

[0119] Table 1.2 Copy number analysis of experimental results in different sturgeon species

[0120] Accuracy determination

[0121] The previously extracted, prepared and diluted standard concentration of Chinese sturgeon DNA nucleic acid solution sample was subjected to digital microdroplet PCR experiment. In order to prevent the occurrence of accidental errors, 4 parallel experiments were performed for each concentration sample, so as to determine the accuracy and minimum concentration detection limit of the microdroplet digital PCR experiment. The results of one experiment are shown in Table 1.3. In the ddPCR experiment of the 14 experimental samples, the number of microdroplets generated in the CS-11 sample experiment was the smallest, which was 11130, and the number of microdroplets generated in the CS-1 sample experiment was the largest, which was 16068. The average number of microdroplets generated in the 14 samples was 13424. Generally, the evaluation standard for judging the experimental results of a sample is whether the total number of microdroplets generated is greater than 10000. If the number of microdroplets generated in the experimental sample is greater than 10000, it means that the experiment is effective and accurate. Otherwise, the experimental results are invalid and unacceptable. The number of microdroplets generated in each of the 14 samples in this experiment is greater than 10000, which means that the microdroplet generation in the ddPCR reaction process of all samples in this experiment is normal, which ensures that the ddPCR experiment can accurately quantify the standard nucleic acid sample, thereby providing a strong guarantee for further in-depth analysis of the experimental results.

[0122] As shown in Figure 3 , it is a one-dimensional droplet scatter plot of the ddPCR experimental results of 14 different concentrations of standard nucleic acid samples. The upper half of the green dots represents the positive microdroplets generated in each reaction, and from the number of green dots, we can find that the number of green dots gradually decreases from left to right until it disappears, which means that the number of positive droplets decreases, indicating that the concentration of the standard nucleic acid sample used in the experiment decreases in order of sample serial number. The lower half is a black dot, which represents the negative droplets generated in each reaction, and from the proportion of black dots, the relative proportion of black droplets increases from left to right, reaching 100%, which means that the last group of experiments has no positive microdroplets, further indicating that the concentration of the standard nucleic acid sample used in the experiment decreases from left to right. In addition, Figure 3 , there is a green line in the middle, which separates the positive droplets above from the negative droplets below, indicating that the positive and negative microdroplets in this ddPCR experiment can be completely distinguished, further indicating that the ddPCR experiment can effectively distinguish between positive and negative reactions, thereby ensuring the accuracy of the digital titration PCR experiment.

[0123] sample Total number of droplets HEX (copies / ul) Number of positive droplets CS-1 16068 4181.00 9037 CS-2 15449 2002.00 5049 CS-3 16419 1080.00 3158 CS-4 12163 590.10 1339 CS-5 14871 289.50 827 CS-6 14741 152.30 437 CS-7 12979 68.70 175 CS-8 14818 39.40 115 CS-9 12734 21.02 52 CS-10 11365 11.60 26 CS-11 11130 0.65 6 CS-12 11566 0.32 4 CS-13 11845 0.12 2 CS-14 11656 0.00 0 NC 11786 0.00 0

[0124] Table 1.3 Copy number statistics table of experimental results of different concentrations of Chinese sturgeon nucleic acid standard liquid

[0125] Minimum detection limit determination

[0126] The minimum detection limit of ddPCR was determined by diluting the standard nucleic acid sample of Chinese sturgeon at equal proportions. The results of ddPCR experiments on the diluted standard nucleic acid sample are shown in Table 1.3. The concentration range of the standard nucleic acid sample CS-1 to CS-13 detected by ddPCR was 0.12 - 4181.00 copies / µl. The initial concentration of the standard nucleic acid sample of Chinese sturgeon CS-1 was 4181.00 copies / µl. The minimum detection concentration was the concentration of the sample CS-12 diluted 4096 times from the initial concentration, and the detection result was 0.12 copies / µl. When the standard sample was diluted for the 14th time (8192 times dilution), no positive droplets were generated in the ddPCR experiment, and the DNA concentration of Chinese sturgeon could not be detected. In addition, the linear analysis of the concentration of Chinese sturgeon DNA sample and the number of dilutions showed that there was a high linear correlation between Log10(standard sample concentration) and log2(dilution ratio) (R²=0.9967, see Figure 4 ). This indicates that the quantitative results of ddPCR have high accuracy and reliability in this concentration range, and are suitable for quantitative detection and analysis of environmental DNA.

[0127] The above merely describes certain exemplary embodiments of the present application by way of illustration, and it is needless to say that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present application. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present application.

Claims

1. An environmental DNA species monitoring method based on Chinese sturgeon, characterized in that, The method comprises the following steps: S1, weighing 500 mg of fresh tail fin tissue or frozen blood sample of Acipenser sinensis; S2, placing the sample in SDS lysis solution, adding appropriate amount of proteinase K and mercaptoethanol, and mixing gently to promote lysis; S3, after lysis is completed, the sample is cooled to room temperature, and then centrifugal treatment is performed; S4, after centrifugation, the supernatant is taken, and chloroform / isopentanol is added for twice extraction; S5, using isopropyl alcohol to precipitate DNA, mixing gently after mixing, and then centrifuging again; S6, discarding the waste liquid, and washing the DNA precipitate with 75% ethanol twice; S7, after the DNA precipitate is dried, 200 μl of EB solution is added for dissolution, and RNAase digestion treatment is performed; S8, purifying the DNA by using OMEGA purification column; S9, after purification is completed, Ampure XP beads are further used for purification; S10, the extracted DNA is detected by Nanodrop, Qubit and electrophoresis method; S11, the mitochondrial whole genome of Acipenser sinensis is determined by combining the second-generation library sequencing technology and the third-generation library sequencing technology, and is compared with the mitochondrial genome of Acipenser sinensis uploaded by NCBI in parallel; S12, by probe design and PCR amplification sequencing, the target DNA sequence of Acipenser sinensis is specifically recognized and combined, and Acipenser sinensis is distinguished from other related species, which is used for subsequent verification analysis experiment.

2. The method according to claim 1, wherein, The second-generation library sequencing technology comprises the following steps: After the DNA sample is detected and confirmed to be qualified, a Covaris ultrasonic crusher is used for random fragmentation treatment; After a series of steps such as end repair, A tail addition, sequencing adapter ligation, purification and PCR amplification, the construction of the library is completed; After the library construction is completed, the concentration is first determined by using Qubit 3.0, and the library is appropriately diluted; The insert size of the library is detected by using Agilent 2100 instrument, and it is ensured that the fragment size meets the expected requirements; BioRAD CFX 96 fluorescence quantitative PCR instrument is used, Bio-RAD KIT iQ SYBR GRN reagent is used, QPCR analysis is performed, and the effective concentration of the library is accurately determined, so as to ensure the quality of the sequencing library; After the library is detected and confirmed to be qualified, different libraries are mixed according to the effective concentration and target data volume requirement; Cluster processing is performed on the flowcell by cBOT, and double-end sequencing program is executed on the NovaSeq 6000 sequencing platform to obtain sequence reads with a length of 150 bp.

3. The method according to claim 1, wherein, The third-generation library sequencing technology comprises the following steps: 2.5 μg of DNA sample qualified by quality inspection is selected, 1× magnetic beads are used for purification treatment, and then 1 μl of sample is taken for quantitative analysis by Qubit; The DNA is subjected to damage repair and end treatment, and the incubation conditions are 20℃ for 10 min, 65℃ for 10 min, and 4℃ for storage; Again, use 1x magnetic bead to purify DNA, and use 61 μl EB buffer to elute, then take 1 μl sample for Qubit quantification; Connect sequencing adapter to DNA sample, incubation condition: 25℃ for 10 min; Use 0.4x magnetic bead to purify DNA, use 25 μl Elution buffer to elute, then take 1 μl sample for Qubit quantification; Prepare sequencing library for machine use; Load the prepared library into R9.4 sequencing chip, and use PromethION sequencer for 48-72 hours of sequencing.

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