A primer for identifying Epinephelus coioides and its application

Abstract

The present invention discloses a primer for identifying Epinephelus coioides and its application, the nucleotide sequence of the primer is shown in SEQ ID NO: 5-6; the application is an application in identifying Epinephelus coioides. The molecular specific marker primer provided by the present invention can perform rapid molecular identification of Epinephelus coioides. The method is simple, accurate, and sensitive, and is an effective auxiliary means for distinguishing Epinephelus coioides by morphological characteristics. Compared with the traditional survey method, which cannot perform effective identification due to incomplete organisms, the detection method of the present invention only needs to collect environmental samples, and can identify whether Epinephelus coioides exists by detecting whether the D-loop region fragment of Epinephelus coioides exists in the environment. It has little damage to the ecosystem, is conducive to the rapid identification of Epinephelus coioides, and has broad application prospects in the protection of germplasm resource diversity and germplasm ecological survey of Epinephelus coioides.

Description

Technical Field

[0001] The present invention relates to the field of fish molecular biology, in particular to a primer for identifying Epinephelus coioides and an application thereof. Background Art

[0002] The coioides grouper (Epinephelus coioides), a member of the order Perciformes and genus Epinephelus, commonly known as the blue grouper, is a valuable marine fish widely farmed along the coasts of Southeast Asia. Due to its delicious and nutritious meat, it holds high economic value. Furthermore, its rapid growth makes it a crucial aquaculture resource in coastal areas.

[0003] Different grouper species share many morphological similarities, making them difficult to distinguish visually, especially at the fry stage. This presents significant challenges in species identification and inconveniences in fry selection and management at hatchery farms. Therefore, developing specific primers for identifying E. coioides populations is crucial for species identification in aquaculture.

[0004] Environmental DNA (eDNA) refers to DNA extracted from environmental samples such as water, soil, or sediment. Free fish DNA in water bodies may come from skin, feces, sperm, and eggs shed or discharged from the fish's body. The use of species-specific molecular markers to detect species distribution and abundance in environmental samples provides a new method for fish species detection, with the advantages of being non-destructive to fish populations, simple and efficient, and highly sensitive. The successful implementation of this technology depends on the detection primers having efficient and specific amplification of a certain fish species. Generally speaking, closely related fish species of the same family or even the same genus are often distributed in the same waters. Commonly used DNA barcode primers are difficult to accurately detect one of the species. Therefore, it is necessary to develop detection primers that can efficiently and specifically amplify molecular markers for the coito-banded grouper in order to achieve the purpose of identifying the coito-banded grouper. Summary of the Invention

[0005] In order to solve the problem in the prior art of lacking detection primers for efficiently and specifically amplifying molecular markers for identifying Epinephelus coioides, the present invention provides a primer for identifying Epinephelus coioides and application thereof.

[0006] The first object of the present invention is to provide a primer for identifying Epinephelus coioides.

[0007] The second object of the present invention is to provide the application of the primer in identifying Epinephelus coioides.

[0008] The third object of the present invention is to provide a method for identifying Epinephelus coioides.

[0009] The fourth object of the present invention is to provide the use of the primers in preparing a kit for identifying Epinephelus coioides.

[0010] A fifth object of the present invention is to provide a test kit for identifying Epinephelus coioides.

[0011] In order to achieve the above object, the present invention is implemented through the following scheme:

[0012] A primer for identifying Epinephelus coioides, wherein the nucleotide sequence of the primer is shown in SEQ ID NO: 5-6.

[0013] The above primers were designed based on the sequence of the D-loop region of the mitochondrial genome of Epinephelus coioides. The D-loop region has a Genbank number of KM377093.1, and the nucleotide sequence of the amplified fragment is shown in SEQ ID NO: 7.

[0014] The application of the above primers in identifying Epinephelus coioides should also be within the scope of protection of the present invention.

[0015] A method for identifying Epinephelus coioides comprises performing PCR amplification using primers with the nucleotide sequences shown in SEQ ID NOs: 5-6; and detecting the PCR amplification products.

[0016] Preferably, in the reaction system of the real-time fluorescence quantitative PCR amplification, the final concentrations of the primers having nucleotide sequences as shown in SEQ ID NOs: 5 to 6 are respectively 0.2 μM to 0.4 μM.

[0017] More preferably, in the reaction system of the real-time fluorescence quantitative PCR amplification, the final concentration of the primers having nucleotide sequences as shown in SEQ ID NOs: 5 to 6 is 0.4 μM respectively.

[0018] Preferably, the reaction procedure of the real-time fluorescence quantitative PCR amplification is: pre-denaturation at 92-98°C for 2-4 min; denaturation at 92-98°C for 25-35 sec; annealing at 54-58°C for 25-35 sec, extension at 70-74°C for 25-35 sec, the number of cycles is 32-38; extension at 70-74°C for 8-12 min.

[0019] More preferably, the reaction procedure of the real-time fluorescence quantitative PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 sec; annealing at 56°C for 30 sec, extension at 72°C for 30 sec, the number of cycles is 35; extension at 72°C for 10 min.

[0020] Preferably, the sample to be tested for identifying Epinephelus coioides is an environmental sample or a nucleic acid sample.

[0021] More preferably, the sample to be tested for identifying Epinephelus coioides is an environmental sample. Before using the primers for PCR amplification, the environmental sample is first filtered through a nitrocellulose filter membrane with a diameter of 45 to 49 mm and a pore size of 0.1 to 0.3 μm, and then DNA is extracted from the filtered filter membrane.

[0022] Further preferably, the sample to be tested for identifying Epinephelus coioides is an environmental sample. Before using the primers for PCR amplification, the environmental sample is first filtered through a nitrocellulose filter membrane with a diameter of 47 mm and a pore size of 0.2 μm, and then DNA is extracted from the filtered filter membrane.

[0023] Further preferably, the environmental sample is a water sample.

[0024] More preferably, the water sample is a seawater sample.

[0025] More preferably, the nucleic acid sample is DNA.

[0026] Preferably, the method for detecting PCR amplification products comprises agarose gel electrophoresis detection and / or sequencing.

[0027] More preferably, the method for detecting PCR amplification products is agarose gel electrophoresis detection.

[0028] The present invention can determine whether the sample to be tested contains Epinephelus coioides by detecting the bands displayed by the agarose gel electrophoresis detection result.

[0029] Preferably, the agarose gel electrophoresis detection method is: preparing 1.5% agarose gel; fully mixing the PCR product with the nucleic acid dye, and then electrophoresing at a voltage of 120V and a current of 200mA for 25min; imaging the gel and observing.

[0030] Preferably, the standard for determining whether Epinephelus coioides exists in the sample to be tested is: if there is a 250bp band, it indicates that Epinephelus coioides exists in the sample to be tested; if there is no 250bp band, it indicates that Epinephelus coioides does not exist in the sample to be tested.

[0031] The use of the above primers in the preparation of a kit for identifying Epinephelus coioides should also be within the scope of protection of the present invention.

[0032] Preferably, the sample to be identified is an environmental sample or a nucleic acid sample.

[0033] More preferably, the sample to be identified is an environmental sample.

[0034] Further preferably, the environmental sample is a water sample.

[0035] More preferably, the water sample is a seawater sample.

[0036] A kit for identifying Epinephelus coioides comprises primers with nucleotide sequences as shown in SEQ ID NOs: 5-6 and PCR reaction reagents.

[0037] Preferably, the method for using the kit comprises the following steps:

[0038] S1. Obtain DNA from the sample to be tested;

[0039] S2. Using the DNA of the test sample as a template, prepare a reaction system using the above primers and PCR reagents, perform PCR amplification, and then detect the PCR amplification product by agarose gel electrophoresis to determine whether the test sample contains Epinephelus coioides;

[0040] The standard for determining whether the sample to be tested contains Epinephelus coioides is as follows: if there is a 250bp band, it indicates that the sample to be tested contains Epinephelus coioides; if there is no 250bp band, it indicates that the sample to be tested contains Epinephelus coioides.

[0041] More preferably, the sample to be tested is an environmental sample. In step S1, the environmental sample is first filtered through a nitrocellulose filter membrane with a diameter of 45 to 49 mm and a pore size of 0.1 to 0.3 μm, and then DNA is extracted from the filtered filter membrane.

[0042] Further preferably, the sample to be tested is an environmental sample. Then, in step S1, the environmental sample is first filtered through a nitrocellulose filter membrane with a diameter of 47 mm and a pore size of 0.2 μm, and then DNA is extracted from the filtered filter membrane.

[0043] Preferably, in the reaction system of the real-time fluorescence quantitative PCR amplification, the final concentrations of the primers having nucleotide sequences as shown in SEQ ID NOs: 5 to 6 are 0.2 μM to 0.4 μM, respectively.

[0044] More preferably, in the reaction system of the real-time fluorescence quantitative PCR amplification, the final concentration of the primers having nucleotide sequences as shown in SEQ ID NOs: 5 to 6 is 0.4 μM respectively.

[0045] Preferably, the reaction procedure of the real-time fluorescence quantitative PCR amplification is: pre-denaturation at 92-98°C for 2-4 min; denaturation at 92-98°C for 25-35 sec; annealing at 54-58°C for 25-35 sec, extension at 70-74°C for 25-35 sec, the number of cycles is 32-38; extension at 70-74°C for 8-12 min.

[0046] More preferably, the reaction procedure of the real-time fluorescence quantitative PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 sec; annealing at 56°C for 30 sec, extension at 72°C for 30 sec, the number of cycles is 35; extension at 72°C for 10 min.

[0047] Preferably, the agarose gel electrophoresis detection method is: preparing 1.5% agarose gel; fully mixing the PCR product with the nucleic acid dye, and then electrophoresing at a voltage of 120V and a current of 200mA for 25min; imaging the gel and observing.

[0048] The application of the above-mentioned kit in identifying Epinephelus coioides should also be within the protection scope of the present invention.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The molecular-specific marker primers provided by the present invention can be used for rapid molecular identification of Epinephelus coioides. The method is simple, accurate, and highly sensitive, and is an effective auxiliary means for distinguishing Epinephelus coioides by morphological characteristics. Compared with traditional survey methods, which cannot effectively identify Epinephelus coioides due to incomplete biological incompleteness, the detection method of the present invention only requires the collection of environmental samples. By detecting the presence of D-loop region fragments of Epinephelus coioides in the environment, the presence of Epinephelus coioides can be identified. This method is less destructive to the ecosystem, facilitates the rapid identification of Epinephelus coioides, and has broad application prospects in the protection of Epinephelus coioides germplasm resource diversity and germplasm ecological surveys. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 1 to 3 are the PCR amplification results of three pairs of primers; M is the DNA standard molecular weight marker; gDNA is the amplification band using the genomic DNA of Epinephelus coioides as a template (positive control); 1 to 3 are the amplification bands using the eDNA of seawater samples as a template.

[0052] Figure 2 are the sequencing and analysis results of the PCR-positive bands; AC are the sequencing results of the positive control; ref is the matching partial sequence of the D-loop region (D-Loop region) of the mitochondrial genome of Epinephelus coioides (Genbank: KM377093.1).

[0053] Figure 3 The figure shows the electrophoresis diagram of genomic DNA of seven fish species; M is the standard DNA molecular weight marker; 1 to 7 are the genomic DNA of coioidal grouper, brown-spotted grouper, saddle-banded grouper, clearwater grouper, leopard gill perch, humpback sea bass and transverse-banded nine-spined sea bass.

[0054] Figure 4: The PCR identification results of seven fish species; M is the DNA standard molecular weight marker; 1 to 7 are PCR amplification bands using genomic DNA of Epinephelus coioides, Epinephelus rufipogon, Epinephelus saddleback, Epinephelus serratus, Epinephelus leopardus, Epinephelus humpback and Epinephelus transverse as templates, respectively.

[0055] Figure 5 The following are the identification results of Epinephelus coioides in the South China Sea; M is the DNA standard molecular weight marker; 1 is the amplified fragment using the genomic DNA of Epinephelus coioides as a template (positive control); 2 to 4 are the amplified fragments using the eDNA of South China Sea seawater samples as templates. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0057] Example 1 Primers for identifying Epinephelus coioides

[0058] 1. Experimental Methods

[0059] 1. Primer design

[0060] Three pairs of primers were designed based on the sequence of the fastest evolving mitochondrial genome hypervariable region (D-Loop region) (SEQ ID NO: 5) of the mitochondrial genome of Epinephelus coioides (Genbank: KM377093.1). The specific sequences are as follows:

[0061] First pair of primers:

[0062] Upstream primer EC-eDNA-04-F: 5′-TGTAATTGTAATGGTTTGCGCAGT-3′ (SEQ ID NO: 1);

[0063] Downstream primer EC-eDNA-04-R: 5′-ACTGAAATATGTTATGTACCACCT-3′ (SEQ ID NO: 2);

[0064] Second pair of primers:

[0065] Upstream primer EC-eDNA-05-F: 5′-GTAATTGTAATGGTTTGCGCAGT-3′ (SEQ ID NO: 3);

[0066] Downstream primer EC-eDNA-05-R: 5′-TACTGAAATATGTTATGTACCACCT-3′ (SEQ ID NO: 4);

[0067] The third pair of primers:

[0068] Upstream primer EC-eDNA-07-F: 5′-TTAGATATATAATGTAATTGTAATGGTTTG-3′ (SEQ ID NO: 5);

[0069] Downstream primer EC-eDNA-07-R: 5′-TCTGGAATAAGTGCTCGGCA-3′ (SEQ ID NO: 6).

[0070] 2. Primer identification

[0071] (1) Extraction of genomic DNA from Epinephelus coioides

[0072] Fin ray samples of Epinephelus coioides were cut and preserved in anhydrous ethanol. Genomic DNA of Epinephelus coioides was extracted from the fin ray samples using a genomic DNA extraction kit (manufacturer: Magen, product number: D3121-03). Subsequent experiments were performed after the samples were qualified after agarose gel electrophoresis.

[0073] (2) Environmental DNA (eDNA) extraction

[0074] At Hailing Island in Yangjiang City, a 3L seawater sample from the bottom of the South China Sea was collected using a water sampler. The 3L seawater was divided into three equal parts and filtered using a nitrocellulose filter membrane with a diameter of 47mm and a pore size of 0.2μm. The seawater was extracted and filtered using a vacuum pump. The filtered filter membranes were frozen at -20℃ for future use.

[0075] The eDNA of the seawater sample was extracted from the filter membrane kept for later use using the Hipure soil DNA kit (manufacturer: Magen) and stored frozen at -20°C.

[0076] (3) PCR amplification

[0077] PCR amplification was performed using the three pairs of primers in this example, using the genomic DNA of Epinephelus coioides (i.e., gDNA, as a positive control) and the eDNA of the seawater sample as templates, respectively.

[0078] The PCR reaction system (total volume 20 μL) was as follows: 2×PCR DS Mix, 10 μL; 10 μM upstream primer, 0.8 μL; 10 μM downstream primer, 0.8 μL; template, 3 μL; ultrapure water, 5.4 μL.

[0079] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 sec; annealing at 56°C for 30 sec, extension at 72°C for 30 sec, cycle number 35; and extension at 72°C for 10 min.

[0080] After the PCR reaction was completed, 3 μL of the PCR product was mixed with TS-GelRed nucleic acid gel dye and subjected to 1.5% agarose gel electrophoresis at a voltage of 120 V and a current of 200 mA for 25 min.

[0081] After electrophoresis, the band amplification was detected by gel imaging system, and the positive bands were sequenced and analyzed.

[0082] 2. Experimental Results

[0083] like Figure 1 As shown, the first pair of primers (EC-eDNA-04-F (SEQ ID NO: 1) and EC-eDNA-04-R (SEQ ID NO: 2)) can amplify the target fragment from the genomic DNA of Epinephelus coioides, but cannot amplify the target fragment from the eDNA of seawater samples; the second pair of primers (EC-eDNA-05-F (SEQ ID NO: 3) and EC-eDNA-05-R (SEQ ID NO: 4)) can amplify the target fragment from the genomic DNA of Epinephelus coioides, but cannot amplify the target fragment from the eDNA of seawater samples; the third pair of primers (EC-eDNA-07-F (SEQ ID NO: 5) and EC-eDNA-07-R (SEQ ID NO: 6)) can amplify the target fragment from both the genomic DNA of Epinephelus coioides and the eDNA of the South China Sea water samples.

[0084] like Figure 2 As shown, the sequencing results showed that the target fragment amplified by PCR using the third pair of primers (EC-eDNA-07-F (SEQ ID NO: 5) and EC-eDNA-07-R (SEQ ID NO: 6)) was 250 bp in length, and the alignment result with the D-loop sequence (SEQ ID NO: 7) of the genomic DNA of Epinephelus coioides showed a 98% match. The 2% deviation between the amplified target fragment and the D-loop sequence (SEQ ID NO: 7) of the genomic DNA of Epinephelus coioides may be due to genetic polymorphism within the population of Epinephelus coioides. The match with the D-loop sequence (SEQ ID NO: 7) of the genomic DNA of Epinephelus coioides was 98% or higher, indicating that the target fragment amplified by PCR was a fragment of the D-loop region of Epinephelus coioides.

[0085] These results demonstrate that the primer pair EC-eDNA-07-F (SEQ ID NO: 5) and EC-eDNA-07-R (SEQ ID NO: 6) have good specificity and sensitivity, can successfully amplify the D-loop region of the genomic DNA of Epinephelus coioides, and can be used to identify Epinephelus coioides, and can effectively distinguish between closely related species and even between different populations of the same species.

[0086] Example 2 Identification of Epinephelus coioides

[0087] 1. Experimental Methods

[0088] 1. Sample DNA Extraction

[0089] Fin ray samples of Epinephelus coioides, Epinephelus rufipogon, Epinephelus saddleback, Epinephelus sepioides, Epinephelus leopardus, Epinephelus humpback and Epinephelus transversalis were cut and preserved in anhydrous ethanol. Genomic DNA of Epinephelus coioides, Epinephelus rufipogon, Epinephelus saddleback, Epinephelus sepioides, Epinephelus leopardus, Epinephelus humpback and Epinephelus transversalis were extracted using a genomic DNA extraction kit (manufacturer: Magen, catalog number: D3121-03). Figure 3 As shown in the figure, the genomic DNA of the seven fish species were tested as qualified by agarose gel electrophoresis and then subjected to subsequent experiments.

[0090] 2. PCR amplification

[0091] The extracted genomic DNA of Epinephelus coioides, Epinephelus rufipogon, Epinephelus saddleback, Epinephelus serratus, Epinephelus leopardus, Epinephelus humpedus, and Epinephelus transversalis were used as templates, and PCR amplification was performed using the primer pairs EC-eDNA-07-F (SEQ ID NO: 5) and EC-eDNA-07-R (SEQ ID NO: 6) of Example 1 of the present invention.

[0092] The PCR reaction system (total volume 20 μL) was as follows: 2× PCR DS Mix, 10 μL; 10 μM upstream primer EC-eDNA-07-F (SEQ ID NO: 5), 0.8 μL; 10 μM downstream primer EC-eDNA-07-R (SEQ ID NO: 6), 0.8 μL; template, 3 μL; ultrapure water, 5.4 μL.

[0093] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 sec; annealing at 56°C for 30 sec, extension at 72°C for 30 sec, cycle number 35; and extension at 72°C for 10 min.

[0094] After the PCR reaction was completed, 3 μL of the PCR product was mixed with TS-GelRed nucleic acid gel dye and subjected to 1.5% agarose gel electrophoresis at a voltage of 120 V and a current of 200 mA for 25 min.

[0095] After electrophoresis, the band amplification was detected using a gel imaging system.

[0096] 2. Experimental Results

[0097] like Figure 4 As shown in the figure, among the genomic DNAs of 7 different fish, only the genomic DNA of Epinephelus coioides could amplify the target band of 250bp, while the target band of the same size was not amplified in the samples of the other six fish closely related to Epinephelus coioides.

[0098] Example 3 A kit for identifying Epinephelus coioides

[0099] 1. Composition

[0100] The method comprises the primer pair EC-eDNA-07-F (SEQ ID NO: 5) and EC-eDNA-07-R (SEQ ID NO: 6) of Example 1 of the present invention, and a PCR reaction reagent, wherein the PCR reaction reagent can be a PCR premix.

[0101] 2. Usage

[0102] (1) Extraction of DNA from test samples

[0103] There are no specific requirements for the extraction method of the test sample DNA. Generally, the test sample DNA can be extracted using conventional laboratory methods (phenol-chloroform extraction method) or commercially available kits.

[0104] When the sample to be tested is an environmental sample (water sample), the water sample can be first filtered using a nitrocellulose filter membrane with a diameter of 47 mm and a pore size of 0.2 μm and a vacuum pump, and then the DNA of the sample to be tested can be extracted from the filtered filter membrane using a commercially available kit.

[0105] (2) PCR reaction system (total volume 20 μL)

[0106] 2×PCR DS Mix, 10 μL; 10 μM upstream primer EC-eDNA-07-F (SEQ ID NO: 5), 0.8 μL; 10 μM downstream primer EC-eDNA-07-R (SEQ ID NO: 6), 0.8 μL; template, 3 μL; ultrapure water, 5.4 μL.

[0107] (3) PCR reaction procedure

[0108] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 sec; annealing at 56°C for 30 sec, extension at 72°C for 30 sec, cycle number 35; extension at 72°C for 10 min.

[0109] (4) Agarose gel electrophoresis detection

[0110] After the PCR reaction was completed, the PCR products were subjected to 1.5% agarose gel electrophoresis at a voltage of 120 V and a current of 200 mA for 25 min.

[0111] After electrophoresis, the band amplification was detected using a gel imaging system.

[0112] (5) Interpretation of results

[0113] When a 250bp band is amplified from the DNA of the sample to be tested, it indicates that Epinephelus coioides is present in the sample to be tested; when a 250bp band is not amplified from the DNA of the sample to be tested, it indicates that Epinephelus coioides is not present in the sample to be tested.

[0114] Example 4 Application of a kit for identifying Epinephelus coioides

[0115] 1. Experimental Methods

[0116] 1. Sample collection

[0117] A 3L seawater sample was collected from the bottom of the South China Sea using a water sampler in the Wanshan Islands, Zhuhai.

[0118] 2. Sample identification

[0119] (1) Use a nitrocellulose filter membrane with a diameter of 47 mm and a pore size of 0.2 μm and a vacuum pump to filter the seawater sample. The filtered filter membrane is frozen and stored at -20°C for later use.

[0120] The eDNA of the seawater sample was extracted from the filter membrane kept for later use using the Hipure soil DNA kit (manufacturer: Magen) and stored frozen at -20°C.

[0121] (2) Using the genomic DNA of Epinephelus coioides obtained in Example 1 (as a positive control) and the eDNA of the seawater sample obtained in this example as templates, PCR reaction and agarose gel electrophoresis detection were performed using the kit of Example 4 of the present invention.

[0122] 2. Experimental Results

[0123] like Figure 5As shown, the kit of Example 4 of the present invention can amplify a target fragment of 250 bp when testing the collected seawater sample, which is consistent with the size of the amplified band of genomic DNA of Epinephelus coioides. This indicates that Epinephelus coioides is present in the South China Sea waters of the Wanshan Islands in Zhuhai City.

[0124] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A primer for identifying Epinephelus coioides using environmental DNA, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NOs: 5-6.

2. Use of the primers according to claim 1 in identifying Epinephelus coioides using environmental DNA.

3. A method for identifying Epinephelus coioides using environmental DNA, characterized in that: Perform PCR amplification using the primers described in claim 1; and detect the PCR amplification product.

4. The method according to claim 3, wherein The PCR amplification is real-time fluorescence quantitative PCR amplification. In the reaction system of the real-time fluorescence quantitative PCR amplification, the final concentrations of the primers having nucleotide sequences as shown in SEQ ID NOs: 5 to 6 are 0.2 μM to 0.4 μM, respectively.

5. The method according to claim 3, wherein The PCR amplification is real-time fluorescence quantitative PCR amplification, and the reaction procedure of the real-time fluorescence quantitative PCR amplification is: pre-denaturation at 92-98°C for 2-4 min; denaturation at 92-98°C for 25-35 sec, annealing at 54-58°C for 25-35 sec, extension at 70-74°C for 25-35 sec, the number of cycles is 32-38; extension at 70-74°C for 8-12 min.

6. The method according to claim 5, wherein The reaction procedure of the real-time fluorescence quantitative PCR amplification was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 30 sec, with a cycle number of 35; and extension at 72°C for 10 min.

7. Use of the primers according to claim 1 in preparing a kit for identifying Epinephelus coioides, characterized in that: The sample to be identified is environmental DNA.

8. A kit for identifying Epinephelus coioides using environmental DNA, characterized in that: Comprising the primer according to claim 1.

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