Primer pair for detecting environment DNA (Deoxyribose Nucleic Acid) of potamogeton pectinatus 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 potamogeton pectinatus and application of the primer pair, the primer pair is selected from any one of the following primers: nucleotide sequences of an upstream primer and a downstream primer of a first primer pair are respectively 5 '-GCGGTGGGCTTGATTTTACC-3' and 5 '-CACATGTACCCGCAGTAGCA-3'; the nucleotide sequences of the upstream primer and the downstream primer of the second primer pair are respectively 5 '-ATGAATGTCTACGCGGGGG-3' and 5 '-TCACATGTACCCGCAGTAGC-3'; the nucleotide sequences of the upstream primer and the downstream primer of the second primer pair are respectively 5 '-ATGAATGTCTACGCGGGG-3'; the nucleotide sequence of the upstream primer of the third primer pair is 5 '-AAAGCACAAGCCGAAACAGG-3', and the nucleotide sequence of the downstream primer of the third primer pair is 5 '-AGCCGTTGTCTCGCAATA-3'. The primer pair provided by the invention can specifically amplify the target DNA of the potamogeton microdentatus, can rapidly and accurately monitor genetic information of the potamogeton microdentatus from a complex environmental sample, avoids non-specific amplification of other types belonging to the same category as the potamogeton microdentatus, and is high in detection sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer pair for detecting environmental DNA of Potamogeton microdentata and an application thereof. Background Art

[0002] Submerged plants absorb nitrogen and phosphorus from the bottom and water through their roots and plant bodies. They have a stronger ability to enrich nitrogen and phosphorus than floating plants, and play an important role in degrading harmful substances in water bodies and repairing water ecology. Submerged plants cover a wide area, mainly including Vallisneria spinulosa, Hydrilla verticillata, Potamogeton sphenodon, Potamogeton spicata, and Ceratophyllum demersum. Among them, Potamogeton sphenodon has a strong ability to remove nitrogen and phosphorus, and can carry out ecological restoration of polluted water bodies. It plays an irreplaceable role in maintaining clean water quality, promoting biodiversity and ecological balance.

[0003] Traditional aquatic plant monitoring methods include direct capture, underwater photography, remote sensing monitoring, and the like. Application No. 202411112770.5 discloses a root sampler and sampling method for submerged plants. Although this direct capture method is feasible, it will damage the growth environment of aquatic plants and is inefficient. Underwater photography is time-consuming and labor-intensive, and the monitoring results are inaccurate. This method cannot be used in turbid water bodies. Remote sensing monitoring methods can generally monitor animals and plants on the water surface, but cannot play their due role in monitoring submerged plants. Therefore, in order to more comprehensively and scientifically evaluate the growth status of Potamogeton odoratum, it is necessary to introduce more comprehensive and advanced detection technologies and methods.

[0004] Environmental DNA technology is an emerging detection method based on molecular biology. It extracts and analyzes trace amounts of free DNA fragments from environmental samples to quickly and accurately monitor the presence and distribution of specific species. The free DNA in these environments will degrade over a period of time, and its presence represents the recent activities of the target species in the area. Since it is not necessary to directly contact or capture the target species to obtain its genetic information, environmental DNA technology has become an important tool for monitoring rare, endangered or cryptic species, especially those whose activities are secretive, whose samples are difficult to obtain, or those that cannot be easily identified by traditional monitoring methods. Therefore, by detecting specific DNA sequences of aquatic plants, including the key species of Potamogeton microdentata, through environmental DNA technology, the diversity of plant species in aquatic ecosystems can be efficiently identified and assessed.

[0005] The invention patent with 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, and combines environmental DNA technology to realize the diversity detection of aquatic plants and improve the detection efficiency; however, the designed primers are universal primers and cannot be used for targeted detection of the presence of Potamogeton odoratum in water bodies. Summary of the invention

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

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

[0008] In a first aspect, the present invention provides a primer pair for detecting environmental DNA of Potamogeton microdentata, characterized in that:

[0009] The primer pair is selected from any one of the following:

[0010] First primer pair:

[0011] The nucleotide sequence of the upstream primer was: 5′-GCGGTGGGCTTGATTTTACC-3′;

[0012] The nucleotide sequence of the downstream primer was: 5′-CACATGTACCCGCAGTAGCA-3′;

[0013] Second primer pair:

[0014] The nucleotide sequence of the upstream primer was: 5′-ATGAATGTCTACGCGGTGGG-3′;

[0015] The nucleotide sequence of the downstream primer was: 5′-TCACATGTACCCGCAGTAGC-3′;

[0016] Third primer pair:

[0017] The nucleotide sequence of the upstream primer was: 5′-AAAGCACAAGCCGAAACAGG-3′;

[0018] The nucleotide sequence of the downstream primer is: 5'-AGCCGTTGTCTCGGCAATAA-3'.

[0019] In the first primer pair, the upstream primer and the downstream primer are respectively located at base 128 to base 147 and base 289 to base 270 of the DNA sequence of Potamogeton microdentata with GENEBANK sequence number KX059535.1;

[0020] In the second primer pair, the upstream primer and the downstream primer are respectively located at base 116 to base 135 and base 290 to base 271 of the Potamogeton microdentata DNA sequence with GENEBANK sequence number KX059535.1;

[0021] In the third primer pair, the upstream primer and the downstream primer are respectively located at the 226th to 245th bases and the 412th to 393rd bases of the Potamogeton microdentata DNA sequence with GENEBANK sequence number KX059535.1.

[0022] In a second aspect, the present invention provides a method for detecting environmental DNA of Potamogeton microdentata, the method comprising:

[0023] S1. Collect water samples in the target waters and extract sample DNA;

[0024] S2. performing qPCR amplification using the primer pair provided in claim 1;

[0025] S3. Analyze the qPCR amplification results to determine whether the water sample contains Potamogeton microdentata.

[0026] In S1, extracting sample DNA includes a pretreatment step and a DNA extraction step;

[0027] 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;

[0028] 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.

[0029] In S2, the reaction system of the 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.

[0030] 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.

[0031] In a third aspect, the present invention provides an application of the aforementioned primer pair for detecting environmental DNA of Potamogeton microdentata in water environment pollution assessment.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The primer pair for detecting environmental DNA of Potamogeton microdentata of the present invention is based on the sequenced Potamogeton microdentata DNA sequence (GENEBANK sequence number is KX059535.1), and is designed for the conserved region in the DNA sequence, and can specifically amplify the target Potamogeton microdentata, avoiding nonspecific amplification of other species of the same genus as Potamogeton microdentata; can quickly and accurately monitor the genetic information of Potamogeton microdentata from complex environmental samples, and has high detection sensitivity, thereby providing strong technical support for the fields of ecological research, species identification and water environment pollution assessment, and can make those Potamogeton microdentata populations that are difficult to monitor by traditional means appear without destroying or fishing the Potamogeton microdentata population, and will not harm submerged vegetation or damage the ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the qPCR amplification curve of the first primer pair for Potamogeton microdentata.

[0035] Figure 2 This is the qPCR melting point curve of the first primer pair for Potamogeton microdentata.

[0036] Figure 3 This is the qPCR amplification curve of the second primer pair for Potamogeton microdentata.

[0037] Figure 4 qPCR melting point curve of the second primer pair for Potamogeton microdentata.

[0038] Figure 5 This is the qPCR amplification curve of the third primer pair for Potamogeton microdentata.

[0039] Figure 6 This is the qPCR melting point curve of the third primer pair for Potamogeton microdentata. DETAILED DESCRIPTION

[0040] The technical scheme 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, it will be understood by those skilled in the art 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 limited by the attached claims and their equivalents.

[0041] Example 1 Design of primer pairs for detecting environmental DNA of Potamogeton microdentata

[0042] Step 1: DNA sequence search and screening

[0043] The NCBI database was used to search for the gene sequence of Potamogeton microdentata; the sequence alignment tool Mega5 was used to align the gene sequence of Potamogeton microdentata with the gene sequences of other Potamogeton microdentata-related species, and finally the Potamogeton microdentata DNA sequence with the GENEBANK sequence number KX059535.1 was selected;

[0044] Step 2: Primer design

[0045] Based on the aforementioned selected DNA sequence of Potamogeton microdentata, the following three primer pairs were designed using the primer design tool Primer Premier 5:

[0046] First primer pair:

[0047] The nucleotide sequence of the upstream primer was: 5′-GCGGTGGGCTTGATTTTACC-3′;

[0048] The nucleotide sequence of the downstream primer was: 5′-CACATGTACCCGCAGTAGCA-3′;

[0049] The upstream primer and the downstream primer are located at the 128th to 147th bases and the 289th to 270th bases of the DNA sequence of Potamogeton microdentata with GENEBANK sequence number KX059535.1, respectively;

[0050] Second primer pair:

[0051] The nucleotide sequence of the upstream primer was: 5′-ATGAATGTCTACGCGGTGGG-3′;

[0052] The nucleotide sequence of the downstream primer was: 5′-TCACATGTACCCGCAGTAGC-3′;

[0053] The upstream primer and the downstream primer are located at the 116th to 135th bases and the 290th to 271th bases of the DNA sequence of Potamogeton microdentata with GENEBANK sequence number KX059535.1, respectively;

[0054] Third primer pair:

[0055] The nucleotide sequence of the upstream primer was: 5′-AAAGCACAAGCCGAAACAGG-3′;

[0056] The nucleotide sequence of the downstream primer is: 5'-AGCCGTTGTCTCGGCAATAA-3'.

[0057] The upstream primer and the downstream primer are respectively located at the 226th to 245th bases and the 412th to 393rd bases of the micro-toothed Potamogeton DNA sequence with GENEBANK sequence number KX059535.1.

[0058] Step 3: Primer specificity verification

[0059] The Primer-Blast tool on the NCBI website was used to verify the specificity of the three primer pairs obtained. The results output by Primer-Blast showed that the three primer pairs could specifically match the DNA sequence fragments of the target species and did not show significant matches with other non-target sequences.

[0060] Example 2 Method for detecting environmental DNA of Potamogeton microdentata

[0061] S1. Collect samples in the target waters and put them into sample tubes;

[0062] S2, performing pretreatment steps and DNA extraction on the sample in sequence to obtain sample DNA;

[0063] The pre-processing step comprises:

[0064] S21. If the sample volume is large, take 0.2-0.5 g of sample from the sample tube and add it to the grinding tube, then add 980 μL of buffer SP and gently vortex to mix; if the sample volume is small, rinse the sample tube with 980 μL of buffer SP (from the FastDNA Spin kit for soil), and then transfer the mixture to the grinding tube;

[0065] S22, add 120 μL of lysis solution LB (from FastDNA Spin kit for soil) to the grinding tube, place the grinding tube in a grinder at 40 Hz for 40 seconds or use Instrument nucleic acid extractor, set the speed to 6.0 and homogenize for 40 seconds;

[0066] S23, centrifuge at 12000 rpm for 5 min, transfer the supernatant to a 2 mL centrifuge tube, add 250 μL of deproteinized solution PL (from FastDNA Spin kit for soil), shake manually 10 times to mix, and place at 4 °C for 5 min;

[0067] S24, centrifuge at 12000 rpm for 3 min at room temperature, then take a maximum of 900 μL of supernatant and transfer it to a 2 mL centrifuge tube;

[0068] S25. Add 1 / 3 of the supernatant volume of binding solution BD (from FastDNA Spin kit for soil). For example, if the supernatant is 900 μL, add 300 μL of binding solution BD. Vortex gently to mix, and place at 4°C for 5 min.

[0069] S26, centrifuge at 12000 rpm for 5 min at room temperature, transfer the supernatant to a 5 mL centrifuge tube, and add 1.5 times the volume of the supernatant buffer RS * (from FastDNA Spin kit for soil), pipette and mix immediately to obtain a mixed solution;

[0070] The DNA extraction step comprises:

[0071] a. Place the DNA adsorption column into a 2 mL collection tube for later use;

[0072] b. Add the pretreated mixed solution to the DNA adsorption column, centrifuge at 12000 rpm for 1 min at room temperature, and discard the waste liquid;

[0073] c. Place the DNA adsorption column back into the collection tube and add 600 μL of rinse solution W. * (from FastDNA Spin kit for soil), centrifuge at 12000 rpm for 30 seconds at room temperature, and discard the rinse solution W * ;

[0074] d. Repeat step c;

[0075] e. Place the DNA adsorption column back into the collection tube and centrifuge the empty column at 12000 rpm at room temperature for 2 minutes to remove the remaining rinse solution W. * ;

[0076] f. Place the DNA adsorption column in a new 1.5mL centrifuge tube, add 30-100μL of elution solution to the center of the filter membrane, and place at room temperature for 3 minutes. Then centrifuge at 12000rpm for 1 minute and collect the filtrate, which is the sample DNA;

[0077] g. Store the sample DNA at -20℃ for short term storage or at -80℃ for long term storage;

[0078] S3, using the above primer pairs using qPCR kit (Roche 480SYBR Green IMaster Kit) was used for qPCR amplification in a qPCR instrument (Ya Rui Biotech MA-6000 real-time fluorescence quantitative PCR instrument); the reaction system of the qPCR amplification was 25 μL, including: 12.5 μL qPCR premix, 1 μL upstream primer, 1 μL downstream primer, 2 μL DNA, 8.5 μL ddH2O; the reaction program of the qPCR amplification was: preheating at 95°C for 10 min; denaturation at 95°C for 15 s; annealing and extension at 60°C for 30 s; 40 cycles;

[0079] S4. Analyze the qPCR amplification results to determine whether the water sample contains Potamogeton microdentata.

[0080] Sensitivity and specificity analysis:

[0081] After sequence alignment, the species names of the same genus and closest sequence to Potamogeton microdentata were obtained as Zostera marina and Lagenaria salsa. Potamogeton microdentata was subjected to control experiments with Zostera marina and Lagenaria salsa, and the Ct value of the qPCR amplification curve and the Tm value of the furnace curve were used to test whether the three designed primer pairs could specifically amplify the target species while avoiding nonspecific amplification with other related species. The Ct value refers to the number of cycles when the fluorescent signal in the amplification reaction first reaches the set threshold. 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 interfered by contamination or inhibitors. Ct values ​​between 15-35 are considered to be effective amplification. Ct values ​​less than 35 indicate that significant fluorescent signals are detected within a fewer number of cycles, which means that a lower concentration of the target gene is detected. Ct values ​​above 35 indicate that more cycles are required during the amplification process to reach the threshold for fluorescent signal detection, which means that there is a mismatch between the designed primer pair and the DNA sequence to be amplified, and thus amplification cannot be performed effectively. Tm values ​​greater than 80°C are considered to be effective amplification. If nonspecific amplification products exist, these nonspecific amplification products may hybridize with the expected amplification products, resulting in a decrease in the melting curve peak, which in turn results in a lower Tm value. The amplification curves and melting furnace curves of the first primer pair for the three micro-toothed Potamogeton qPCR were obtained as follows: Figure 1 , Figure 2The Ct value and Tm value of the first primer pair are shown in Table 1; the qPCR amplification curves and melting furnace curves of the second primer pair for three samples of Potamogeton microdentata are shown in Figure 3 , Figure 4 The Ct value and Tm value of the second primer pair are shown in Table 2. The qPCR amplification curves and melting furnace curves of the third primer pair for three samples of Potamogeton microdentata are shown in Table 3. Figure 3 , Figure 4 As shown, the Ct value and Tm value of the third primer pair are shown in Table 3:

[0082] Table 1 Ct value and Tm value of the first primer pair

[0083] Sample name Ct value Tm value Potamogeton microdentata 1 15.51 81.39 Potamogeton microdentata 2 15.64 81.39 Potamogeton microdentata 3 16.28 81.39 Zostera marina - - Sea lettuce - -

[0084] Table 2 Ct value and Tm value of the second primer pair

[0085] Sample name Ct value Tm value Potamogeton microdentata 1 15.5 81.78 Potamogeton microdentata 2 15.79 81.78 Potamogeton microdentata 3 15.64 81.78 Zostera marina - - Sea lettuce - -

[0086] Table 3 Ct value and Tm value of the third primer pair

[0087] Sample name Ct value Tm value Potamogeton microdentata 1 16.73 82.54 Potamogeton microdentata 2 16.97 82.54 Potamogeton microdentata 3 16.74 82.54 Zostera marina - - Sea lettuce - -

[0088] From Table 1 to Table 3, it can be seen that the Ct value of qPCR amplification of Potamogeton micro-toothed using the three primer pairs proposed in the present invention is stable at about 15-16, which indicates that the amplification efficiency is good, the template concentration is moderate, and the experimental conditions are stable. At the same time, the Tm values ​​of qPCR amplification of Potamogeton micro-toothed using the three primer pairs proposed in the present invention are stable at 82.54, 81.39, and 80.63, which indicates that the three primer pairs proposed in the present invention are relatively stable in combination with the target DNA fragment of Potamogeton micro-toothed, further confirming the specificity and reliability of the amplified product. However, the qPCR amplification of Zostera marina and Sea lettuce, which are of the same genus as Potamogeton micro-toothed using the three primer pairs proposed in the present invention, did not show signs of amplification, and the corresponding Ct value and Tm value could not be obtained, which indicates that it is impossible to specifically amplify other Potamogeton micro-toothed species of the same genus except the target species. The above results can prove that the three primer pairs proposed in the present invention all have good amplification specificity.

Claims

1. A primer pair for detecting environmental DNA of Potamogeton microdentata, 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′-GCGGTGGGCTTGATTTTACC-3′; The nucleotide sequence of the downstream primer was: 5′-CACATGTACCCGCAGTAGCA-3′; Second primer pair: The nucleotide sequence of the upstream primer was: 5′-ATGAATGTCTACGCGGTGGG-3′; The nucleotide sequence of the downstream primer was: 5′-TCACATGTACCCGCAGTAGC-3′; Third primer pair: The nucleotide sequence of the upstream primer was: 5′-AAAGCACAAGCCGAAACAGG-3′; The nucleotide sequence of the downstream primer is: 5'-AGCCGTTGTCTCGGCAATAA-3'.

2. A primer pair for detecting environmental DNA of Potamogeton microdentata according to claim 1, characterized in that: In the first primer pair, the upstream primer and the downstream primer are respectively located at base 128 to base 147 and base 289 to base 270 of the DNA sequence of Potamogeton microdentata with GENEBANK sequence number KX059535.1; In the second primer pair, the upstream primer and the downstream primer are respectively located at base 116 to base 135 and base 290 to base 271 of the Potamogeton microdentata DNA sequence with GENEBANK sequence number KX059535.1; In the third primer pair, the upstream primer and the downstream primer are respectively located at the 226th to 245th bases and the 412th to 393rd bases of the Potamogeton microdentata DNA sequence with GENEBANK sequence number KX059535.

1.

3. A method for detecting environmental DNA of Potamogeton microdentata, characterized in that: The method comprises: S1. Collect water 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 Potamogeton microdentata.

4. A method for detecting environmental DNA of Potamogeton microdentata according to claim 3, characterized in that: In S1, extracting sample 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 environmental DNA of Potamogeton microdentata according to claim 4, characterized in that: In S2, the reaction system of the 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 environmental DNA of Potamogeton microdentata 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 environmental DNA of Potamogeton microdentata according to any one of claims 1 or 2 in water environment pollution assessment.

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