Primer pair for detecting environment DNA of vallisneria spinulosa and application of primer pair

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a primer pair for detecting environment DNA of vallisneria spinulosa and application of the primer pair, the primer pair is selected from any one of the following primer pairs: a first primer pair: an upstream primer nucleotide sequence is 5 '-CTTGCAGCATTCCGAGTCA-3', and a downstream primer nucleotide sequence is 5 '-GCCCATAGTCCACAGTT-3'; according to the second primer pair, the nucleotide sequence of the upstream primer is 5 '-ATCTTGCAGCATTCCGAGT-3', and the nucleotide sequence of the downstream primer is 5 '-AAGCCATCAGTCCACACAG-3'. The primer pair provided by the invention can specifically amplify target DNA of the vallisneria spinulosa, can rapidly and accurately monitor genetic information of the vallisneria spinulosa from a complex environmental sample, avoids non-specific amplification of other types of vallisneria spinulosa, and is high in detection sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a primer pair for detecting the environmental DNA of Vallisneria spinulosa and its application. Background Art

[0002] As an important part of the aquatic ecosystem, Vallisneria spinulosa plays an irreplaceable role in maintaining water quality cleanliness, promoting biodiversity and ecological balance. They can absorb nutrients, purify water quality, provide habitats and affect water flow dynamics. At the same time, Vallisneria spinulosa is highly sensitive to chemicals, so it is often used as an indicator species for water environmental pollution. However, Vallisneria spinulosa usually grows in relatively shallow waters, and its growth state is easily affected by various factors such as water flow, light and substrate, making it difficult for conventional monitoring methods to accurately reflect its true situation.

[0003] Traditional monitoring methods for aquatic plants include direct fishing method, underwater photography method, remote sensing monitoring method, etc. Application No. 202411112770.5 discloses a root sampler and sampling method for submerged plants. Although this direct fishing method is feasible, it will damage Myriophyllum verticillatum, Vallisneria natans and other irrelevant aquatic plants, and the efficiency is low. The underwater photography method is time-consuming and laborious, and the monitoring results are not accurate. This method cannot be used in turbid water bodies. The remote sensing monitoring method can generally monitor animals and plants on the water surface and cannot play its due role in monitoring submerged plants. Therefore, in order to more comprehensively and scientifically evaluate the growth status of Vallisneria spinulosa, more comprehensive and advanced detection technologies and methods need to be introduced.

[0004] Environmental DNA technology is an emerging detection method based on molecular biology. By extracting and analyzing trace free DNA fragments from environmental samples, it can quickly and accurately monitor the presence and distribution of specific species. These free DNAs in the environment will degrade within a certain period of time, and their presence represents the recent activities of the target species in this area. Since genetic information of the target species can be obtained without directly contacting or capturing it, environmental DNA technology has become an important tool for monitoring rare, endangered or cryptic species, especially those with secretive activities, difficult-to-obtain samples or species that cannot be easily identified by traditional monitoring means. Therefore, by detecting specific DNA sequences of aquatic plants, including key species of Vallisneria spinulosa, through environmental DNA technology, the plant species diversity in the aquatic ecosystem can be efficiently identified and evaluated.

[0005] The invention patent with the application number 202011048150.1 provides a method for detecting the diversity of large aquatic plants based on environmental DNA technology. This method designs universal primers for aquatic plants based on the Rbcl gene, combines environmental DNA technology to achieve the detection of the diversity of aquatic plants, and improves the detection efficiency. However, the designed primers are universal primers and cannot be used for targeted detection of the presence of Vallisneria spinulosa in water bodies. Summary of the Invention

[0006] The object of the present invention is to provide a primer pair with strong specificity for detecting environmental DNA of Vallisneria spinulosa and its application in view of the above problems existing in the prior art.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] In the first aspect, the present invention provides a primer pair for detecting environmental DNA of Vallisneria spinulosa, and the primer pair is selected from any one of the following:

[0009] The first primer pair:

[0010] The nucleotide sequence of the upstream primer is: 5'-CTTGGCAGCATTCCGAGTCA-3';

[0011] The nucleotide sequence of the downstream primer is: 5'-GCCCATCAGTCCACACAGTT-3';

[0012] The second primer pair:

[0013] The nucleotide sequence of the upstream primer is: 5'-ATCTTGGCAGCATTCCGAGT-3';

[0014] The nucleotide sequence of the downstream primer is: 5'-AAGCCCATCAGTCCACACAG-3'.

[0015] In the first primer pair, the upstream primer and the downstream primer are respectively located at the 63rd base to the 82nd base and the 175th base to the 156th base of the Vallisneria spinulosa DNA sequence with the GENEBANK serial number JF944749.1; in the second primer pair, the upstream primer and the downstream primer are respectively located at the 61st base to the 80th base and the 177th base to the 158th base of the Vallisneria spinulosa DNA sequence with the GENEBANK serial number JF944749.1.

[0016] In the second aspect, the present invention provides a method for detecting environmental DNA of Vallisneria spinulosa, and the method includes:

[0017] S1. Collect samples in the target water area and extract the sample DNA;

[0018] S2. Perform qPCR amplification using the aforementioned primer pair;

[0019] S3. Analyze the qPCR amplification results to determine whether the water sample contains *Vallisneria spinulosa*.

[0020] In S1, the extraction of environmental DNA includes a pretreatment step and a DNA extraction step;

[0021] The pretreatment step includes: placing the sample in a grinding tube, first adding buffer SP and then adding lysis buffer LB, placing the grinding tube in a grinder or nucleic acid extractor to lyse out DNA, then centrifuging to obtain the supernatant, adding protein removal solution PL and mixing well, standing for a certain time and then centrifuging again to obtain the supernatant, adding binding solution BD and mixing well, standing for a certain time and then centrifuging again to obtain the supernatant, and adding buffer RS * to obtain a mixed solution for standby;

[0022] The DNA extraction step includes: adding the mixed solution obtained in the pretreatment step to a DNA adsorption column, centrifuging and discarding the waste liquid, rinsing the DNA adsorption column twice with washing solution W * and centrifuging to discard the remaining washing solution W * , then adding elution buffer to the DNA adsorption column, standing at room temperature and then centrifuging to collect the filtrate to obtain sample DNA.

[0023] In S2, the reaction system for qPCR amplification is calculated based on 25 μL and includes: 12.5 μL of qPCR premix, 1 μL of upstream primer, 1 μL of downstream primer, 2 μL of DNA, and 8.5 μL of ddH2O.

[0024] In S2, the reaction program for qPCR amplification is: preheating at 95 °C for 10 min; denaturing at 95 °C for 15 s; annealing and extending at 60 °C for 30 s; 40 cycles.

[0025] In the third aspect, the present invention provides the application of the aforementioned primer pair for detecting the environmental DNA of *Vallisneria spinulosa* in the assessment of water environmental pollution.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] The primer pair for detecting the environmental DNA of Vallisneria spinulosa described in the present invention is designed based on the sequenced DNA sequence of Vallisneria spinulosa (GENEBANK accession number: JF944749.1), and is designed for the conserved region in this DNA sequence, which can specifically amplify the target Vallisneria spinulosa and avoid non-specific amplification with other species in the same genus as Vallisneria spinulosa; it can quickly and accurately monitor the genetic information of Vallisneria spinulosa from complex environmental samples with high detection sensitivity, thus providing strong technical support for fields such as ecological research, species identification, and water environmental pollution assessment. Without damaging or fishing the Vallisneria spinulosa population, the Vallisneria spinulosa population that is difficult to monitor by traditional means can be revealed, and the submerged vegetation will not be damaged or the ecological environment will not be destroyed. Description of the Drawings

[0028] Figure 1 It is the qPCR amplification curve of the first primer pair for Vallisneria spinulosa.

[0029] Figure 2 It is the qPCR melting curve of the first primer pair for Vallisneria spinulosa.

[0030] Figure 3 It is the qPCR amplification curve of the second primer pair for Vallisneria spinulosa.

[0031] Figure 4 It is the qPCR melting curve of the second primer pair for Vallisneria spinulosa. Detailed Embodiments

[0032] The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, those of ordinary skill in the technical field to which the present invention belongs will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0033] Embodiment 1 Design of the Primer Pair for Detecting the Environmental DNA of Vallisneria spinulosa

[0034] Step 1. Searching and Screening of DNA Sequences

[0035] Use the NCBI database to search for the gene sequence of Vallisneria spinulosa; use the sequence alignment tool Mega5 to align the gene sequence of Vallisneria spinulosa with the gene sequences of other related species of Vallisneria spinulosa, and finally select the Vallisneria spinulosa DNA sequence with the GENEBANK accession number JF944749.1;

[0036] Step 2. Primer Design

[0037] Based on the aforementioned selected Vallisneria spinulosa DNA sequence, the following two primer pairs were designed using the primer design tool Primer Premier 5:

[0038] The first primer pair:

[0039] The nucleotide sequence of the upstream primer is: 5'-CTTGGCAGCATTCCGAGTCA-3';

[0040] The nucleotide sequence of the downstream primer is: 5'-GCCCATCAGTCCACACAGTT-3';

[0041] This upstream primer and downstream primer are located at the 63rd to 82nd bases and the 175th to 156th bases of the Vallisneria spinulosa DNA sequence with the GENEBANK accession number JF944749.1, respectively;

[0042] The second primer pair:

[0043] The nucleotide sequence of the upstream primer is: 5'-ATCTTGGCAGCATTCCGAGT-3';

[0044] The nucleotide sequence of the downstream primer is: 5'-AAGCCCATCAGTCCACACAG-3';

[0045] This upstream primer and downstream primer are located at the 61st to 80th bases and the 177th to 158th bases of the Vallisneria spinulosa DNA sequence with the GENEBANK accession number JF944749.1, respectively;

[0046] Step 3. Primer specificity verification

[0047] The Primer-Blast tool on the NCBI website was used to verify the specificity of the two obtained primer pairs. The results output by Primer-Blast showed that these two primer pairs could specifically match the DNA sequence fragments of the target species and did not show significant matches with other non-target sequences.

[0048] Example 2 Method for detecting environmental DNA of Vallisneria spinulosa

[0049] S1. Collect samples in the target water area into the sample tube;

[0050] S2. Perform pretreatment steps and DNA extraction on the samples in sequence to obtain sample DNA;

[0051] The pretreatment steps include:

[0052] S21. If the sample volume is large, take 0.2 - 0.5 g of the sample from the sample tube and add it to the grinding tube, then add 980 μL of buffer SP and gently vortex to mix evenly; if the sample volume is small, rinse the sample tube with 980 μL of buffer SP (from the FastDNA Spin kit for soil kit), and then transfer the mixture to the grinding tube;

[0053] S22. Add 120 μL of lysis buffer LB (from the FastDNA Spin kit for soil kit) to the grinding tube, place the grinding tube in the grinder and process it at 40 Hz for 40 seconds or use the Instrument nucleic acid extractor, set the speed to 6.0 and homogenize for 40 seconds;

[0054] S23. Centrifuge at 12000 rpm for 5 min, then transfer the supernatant to a 2 mL centrifuge tube, add 250 μL of protein removal solution PL (from the FastDNA Spin kit for soil kit), manually shake 10 times to mix evenly, and place it at 4 °C for 5 min;

[0055] S24. Centrifuge at 12000 rpm at room temperature for 3 min, then take up to 900 μL of the supernatant and transfer it to a 2 mL centrifuge tube;

[0056] S25. Add binding solution BD (from the FastDNA Spin kit for soil kit) with a volume of 1 / 3 of the supernatant volume. For example, if the supernatant is 900 μL, then add 300 μL of binding solution BD; gently vortex to mix evenly, and place it at 4 °C for 5 min.

[0057] S26. Centrifuge at 12000 rpm at room temperature for 5 min, then transfer the supernatant to a 5 mL centrifuge tube, add buffer RS * (from the FastDNA Spin kit for soil kit), immediately pipette to mix evenly to obtain a mixed solution;

[0058] The DNA extraction steps include:

[0059] a. Set the DNA adsorption column into a 2 mL collection tube for standby;

[0060] b. Add the pretreated mixed solution to the DNA adsorption column, centrifuge at 12000 rpm at room temperature for 1 min, and discard the waste liquid;

[0061] c. Put the DNA adsorption column back into the collection tube, add 600 μL of washing solution W *(From the FastDNA Spin kit for soil kit), centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the wash buffer W * ;

[0062] d. Repeat step c once;

[0063] e. Put the DNA adsorption column back into the collection tube, and centrifuge the empty column at 12,000 rpm for 2 min at room temperature to remove the residual wash buffer W * ;

[0064] f. Put the DNA adsorption column into a new 1.5 mL centrifuge tube, add 30 - 100 μL of elution buffer to the center of the filter membrane, and let it stand at room temperature for 3 min. Then centrifuge at 12,000 rpm for 1 min to collect the filtrate, which is the sample DNA;

[0065] g. Store the sample DNA at -20 °C for short-term or at -80 °C for long-term;

[0066] S3. Use the aforementioned primer pair with a qPCR kit (Roche 480 SYBR Green I Master kit) in a qPCR instrument (Yare Bio MA-6000 type real-time fluorescence quantitative PCR instrument) for qPCR amplification; the reaction system for the qPCR amplification is 25 μL in total, including: 12.5 μL of qPCR premix, 1 μL of upstream primer, 1 μL of downstream primer, 2 μL of DNA, and 8.5 μL of ddH2O; the reaction program for the qPCR amplification is: preheat at 95 °C for 10 min; denature at 95 °C for 15 s; anneal and extend at 60 °C for 30 s; 40 cycles;

[0067] S4. Analyze the qPCR amplification results to determine whether the water sample contains Vallisneria spinulosa.

[0068] Sensitivity and specificity analysis:

[0069] After sequence alignment, the species with the closest sequence and belonging to the same genus as *Vallisneria spinulosa* were identified as *Vallisneria natans* and *Vallisneria asiatica*. A control experiment was conducted with *Vallisneria spinulosa*, *Vallisneria natans*, and *Vallisneria asiatica*. The Ct values of the qPCR amplification curves and the Tm values of the melting curves were used to test whether the two designed primer pairs could specifically amplify the target species while avoiding non-specific amplification with other related species. The Ct value refers to the number of cycles when the fluorescence signal first reaches the set threshold in the amplification reaction. A stable Ct value indicates that the primer design is reasonable and can effectively amplify the target sequence, and the experimental operation process is not contaminated or interfered by inhibitors. A Ct value between 15 - 35 is considered effective amplification. A Ct value less than 35 indicates that a significant fluorescence signal is detected within a smaller number of cycles, meaning a lower concentration of the target gene is detected. A Ct value above 35 indicates that more cycles are required during the amplification process to reach the fluorescence signal detection threshold, meaning there is a mismatch between the designed primer pair and the DNA sequence to be amplified, resulting in ineffective amplification. A Tm value greater than 80 °C is considered effective amplification. If there are non-specific amplification products, these non-specific amplification products may hybridize with the expected amplification products, causing a decrease in the peak of the melting curve and thus a lower Tm value. The qPCR amplification curves and melting curves of the first primer pair for the three samples of *Vallisneria spinulosa* are respectively as shown in Figure 1 , Figure 2 . The Ct values and Tm values of the first primer pair are shown in Table 1; The qPCR amplification curves and melting curves of the second primer pair for the three samples of *Vallisneria spinulosa* are respectively as shown in Figure 3 , Figure 4 . The Ct values and Tm values of the second primer pair are shown in Table 2:

[0070] Table 1 Ct values and Tm values of the first primer pair

[0071]

[0072]

[0073] Table 2 Ct values and Tm values of the second primer pair

[0074] Sample Name Ct Value Tm Value Vallisneria spinulosa 1 29.91 85.6 Vallisneria spinulosa 2 29.39 85.6 Vallisneria spinulosa 3 29.98 85.6 Vallisneria natans - - Vallisneria asiatica - -

[0075] As can be seen from Table 1 and Table 2, the Ct values of qPCR amplification of three samples of Vallisneria spinulosa using the first primer pair and the second primer pair are stable between 29 and 30, indicating good amplification efficiency, moderate template concentration, and stable experimental conditions. At the same time, the Tm values of qPCR amplification of three samples of Vallisneria spinulosa using the first primer pair and the second primer pair are stable around 85.6, indicating that the primer pairs bind relatively stably to the target DNA fragments of Vallisneria spinulosa. This stability stems from the high complementarity between the primer sequences and the target DNA sequences, enabling the primers to bind firmly to the target DNA fragments during the high-temperature denaturation stage of the PCR reaction, thereby effectively guiding the extension of DNA strands during the subsequent annealing and extension stages, further confirming the specificity and reliability of the amplification products. However, when using the first primer pair and the second primer pair to perform qPCR amplification on other species within the same genus as Vallisneria spinulosa, no amplification signs were observed, and corresponding Ct values and Tm values could not be obtained, indicating that it is impossible to specifically amplify other species within the same genus as Vallisneria spinulosa except for the target species. The above results prove that both the first primer pair and the second primer pair described in the present invention have good amplification specificity.

Claims

1. A primer pair for detecting Vallisneria spinulosa environmental DNA, characterized in that: The primer pair is selected from any one of the following: First primer pair: The nucleotide sequence of the upstream primer was: 5′-CTTGGCAGCATTCCGAGTCA-3′; The nucleotide sequence of the downstream primer was: 5′-GCCCATCAGTCCACACAGTT-3′; Second primer pair: The nucleotide sequence of the upstream primer was: 5′-ATCTTGGCAGCATTCCGAGT-3′; The nucleotide sequence of the downstream primer is: 5'-AAGCCCATCAGTCCACACAG-3'.

2. A primer pair for detecting Vallisneria spinulosa environmental DNA according to claim 1, characterized in that: In the first primer pair, the upstream primer and the downstream primer are respectively located at the 63rd to 82nd base and the 175th to 156th base of the Vallisneria spinulosa DNA sequence with the GENEBANK sequence number JF944749.1; in the second primer pair, the upstream primer and the downstream primer are respectively located at the 61st to 80th base and the 177th to 158th base of the Vallisneria spinulosa DNA sequence with the GENEBANK sequence number JF944749.

1.

3. A method for detecting Vallisneria spinulosa environmental DNA, characterized in that: The method comprises: S1. Collect samples in the target waters and extract sample DNA; S2. performing qPCR amplification using the primer pair provided in claim 1; S3. Analyze the qPCR amplification results to determine whether the water sample contains Vallisneria spinulosa.

4. A method for detecting Vallisneria spinulosa environmental DNA according to claim 3, characterized in that: In S1, the extraction of environmental DNA includes a pretreatment step and a DNA extraction step; The pretreatment step includes: placing the sample in a grinding tube, adding a buffer solution SP and then a lysis solution LB, placing the grinding tube in a grinder or a nucleic acid extractor to lyse the DNA, then centrifuging to obtain the supernatant, adding a deproteinization solution PL to mix, standing for a certain period of time, centrifuging again to obtain the supernatant, adding a binding solution BD to mix, standing for a certain period of time, centrifuging again to obtain the supernatant, adding a buffer solution RS to obtain the supernatant, and then adding a buffer solution LS to obtain the supernatant. * The mixed solution is then obtained for standby use; The DNA extraction step comprises: adding the mixed solution obtained in the pretreatment step to a DNA adsorption column, discarding the waste liquid after centrifugation, and washing with a rinse solution W * Rinse the DNA adsorption column twice, centrifuge and discard the remaining rinsing solution W * , then add the elution solution to the DNA adsorption column, let it stand at room temperature and then centrifuge to collect the filtrate to obtain the sample DNA.

5. A method for detecting Vallisneria spinulosa environmental DNA according to claim 4, characterized in that: In S2, the reaction system for qPCR amplification is 25 μL, including: 12.5 μL qPCR premix, 1 μL upstream primer, 1 μL downstream primer, 2 μL DNA, and 8.5 μL ddH2O.

6. A method for detecting Vallisneria spinulosa environmental DNA according to claim 3, characterized in that: In S2, the reaction procedure of the qPCR amplification is: preheating at 95°C for 10 min; denaturation at 95°C for 15 s; annealing and extension at 60°C for 30 s; and 40 cycles.

7. Use of the primer pair for detecting Vallisneria spinulosa environmental DNA according to any one of claims 1 or 2 in water environment pollution assessment.

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