Primer group, kit and method for rapidly identifying components of pink salmon

Abstract

The invention discloses a primer group, a kit and a method for rapidly identifying a pink salmon component, belongs to the technical field of nucleic acid detection of marine fish meat products, and particularly relates to a method for rapidly identifying the pink salmon component by using an LAMP (Loop-Mediated Isothermal Amplification) technology. The primer group comprises a pair of outer primers, a pair of inner primers and a loop primer, and the kit comprises the primer group and LAMP amplification reaction liquid. According to the present invention, the sample DNA is only required to be added into the LAMP amplification reaction liquid, the visual qualitative detection can be achieved through the simple constant temperature device, the operation is simple, the reaction time is only 1 h, the cover does not need to be opened after the reaction is completed, the experiment result can be obtained by observing the color change of the reaction liquid, the high concentration DNA aerosol pollution is effectively prevented, and the detection result is accurate. When the color of the reaction liquid is yellow, the detected sample is determined to be positive, and when the color of the reaction liquid is red, the detected sample is determined to be negative. The method also has the advantages of strong specificity, high sensitivity, good repeatability and low cost, the genome DNA sensitivity can reach 1 ng, the mass fraction sensitivity can reach 1%, the supervision cost is greatly saved, the supervision efficiency is improved, and the method is suitable for rapid detection of vast basic levels and field assurance with capital shortage.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection of marine fish products, and specifically relates to a method for rapidly identifying pink salmon components using LAMP technology, a specific nucleotide primer set used in the method, and a kit containing the primer set. Background Art

[0002] Salmon is a world-renowned, high-value commercial fish, nutritious and delicious, making it a favorite among consumers. Salmon is not a single fish, but rather a group of fish that grow in the ocean and, upon reaching sexual maturity, migrate to freshwater to spawn. These include Atlantic salmon (Salmo salar), King salmon (Oncorhynchus tshawytscha), Coho salmon (Oncorhynchus kisutch), Pink salmon (Oncorhynchus gorbuscha), Sockeye salmon (Oncorhynchus nerka), and Oxtail salmon (Oncorhynchus keta). Because these fish have varying prices and bear striking similarities in texture and appearance, some unscrupulous vendors are passing off cheaper salmon or rainbow trout as pricier King salmon, harming the legitimate rights and interests of consumers.

[0003] Currently, real-time fluorescence PCR technology has become the most common method for identifying fish-derived ingredients. This method requires heating and cooling the sample and detecting fluorescent signals during the experiment, which requires very strict temperature control. The instrumentation used is complex and precise, making it relatively expensive. The probes used in the experiment are also expensive, resulting in high costs.

[0004] As a novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP) overcomes the shortcomings of traditional PCR and real-time fluorescence PCR detection methods, such as repeated temperature increases and decreases, precise instrumentation, high costs, and relatively long detection cycles. It enables continuous and rapid amplification under constant temperature conditions. The method for rapidly identifying pink salmon components based on LAMP isothermal amplification technology is highly specific, sensitive, simple to operate, requires no opening of the container, and is time-efficient, requiring only one hour to complete. It eliminates the need for complex, repeated temperature increases and precise fluorescence signal recognition equipment, requiring only a simple constant temperature device. The test can be completed by visually observing color changes, demonstrating promising application prospects for identifying salmon authenticity and rapidly detecting fish products. Summary of the Invention

[0005] To solve the above practical problems, the present invention aims to analyze the genetic background of pink salmon, design specific nucleotide primers based on the pink salmon ATP6 gene sequence, and use LAMP isothermal amplification technology to quickly identify pink salmon components.

[0006] The present invention is achieved through the following technical solutions.

[0007] The primer set for rapidly identifying pink salmon components includes: an outer primer F3: 5'-AGATGCGACCTAATACGAAAT-3' (SEQ ID No. 1); an outer primer B3: 5'-CAATAAGCCAAACACGTGAT-3' (SEQ ID No. 2); an inner primer FIP: 5'-CGGTTTAATGTAGTAAGAACCGACCCCCTGACCATCAATAAGAGT-3' (SEQ ID No. 3); an inner primer BIP: 5'-CTGATGCTGAAAGTTGGTGGGTTGTCTGCAACTGATTAATTGC-3' (SEQ ID No. 4); and a loop primer LB: 5'-CCCGTGTTAGCTGGAGGTTTG-3' (SEQ ID No. 5).

[0008] The kit for rapidly identifying pink salmon components of the present invention comprises the above-mentioned primer set. Further, the kit also comprises 23 μL of LAMP amplification reaction solution, comprising the following components: LAMP premix (2×): 12.5 μL, 0.1 μL each of SEQ ID No. 1 (100 μmol / L) and SEQ ID No. 2 (100 μmol / L), 0.4 μL each of SEQ ID No. 3 (100 μmol / L) and SEQ ID No. 4 (100 μmol / L), 0.2 μL of SEQ ID No. 5 (100 μmol / L), supplemented with ddH2O to 23 μL.

[0009] The method for identifying pink salmon components of the present invention comprises the following steps.

[0010] (1) Extract sample DNA.

[0011] (2) Add 2 μL of the extracted sample DNA to the LAMP amplification reaction solution of the kit.

[0012] (3) In a constant temperature device: 61°C; 60 min.

[0013] The kit also includes positive control, negative control and blank control. Positive control: 2 μL of pink salmon genomic extract is used as a template and added to the LAMP amplification reaction solution (23 μL) of the kit; negative control: 2 μL of genomic extract of other species without positive DNA components is used as a template; blank control: the same volume of ddH2O is used as a template.

[0014] The quality control standard for judging the amplification results is: if any of the following conditions is not met, the experiment is considered invalid: (a) blank control: the reaction solution color is red; (b) negative control: the reaction solution color is red; (c) positive control: the reaction solution color is yellow.

[0015] Result determination: When the reaction solution is yellow, the sample is determined to be positive. When the reaction solution is red, the sample is determined to be negative.

[0016] In the LAMP detection method described above, the sample DNA can be extracted using a commercially available DNA extraction kit, or using the CTAB method, alkaline lysis method, etc.

[0017] The present invention has the following beneficial effects: (1) Simple and efficient operation, avoiding aerosol pollution; the entire reaction process can be completed in only 60 minutes, and there is no need to perform electrophoresis, purification, sequencing, and comparison on the nucleic acid amplification products. There is no need to open the lid after the reaction is completed, and only the color change of the reaction solution needs to be observed with the naked eye, which has the advantages of preventing pollution and short time consumption; (2) High cost performance and low cost; no sophisticated and expensive repeated heating and cooling instruments and fluorescent signal receiving devices are required, and only the color change of the reaction solution needs to be observed with the naked eye. The primer set does not need to be connected to expensive probes and fluorescent groups, which greatly saves regulatory costs and improves regulatory efficiency. It is suitable for rapid detection of grassroots and on-site guarantees with insufficient funds; (3) High specificity; the specific primer set of the present invention contains 5 primers. Any region in the template that cannot match the above primers cannot be amplified, and other species of the same genus cannot be amplified; (4) High sensitivity, and the sensitivity of genomic DNA can reach 1 ng, and the mass fraction sensitivity can reach 1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the specific test result; Figure 1 The numbers in the chart represent: 1: King salmon; 2: Coho salmon; 3: Pink salmon; 4: Sockeye salmon; 5: Chub salmon; 6: Atlantic salmon; 7: Rainbow trout; 8: Atlantic mackerel; 9: Japanese mackerel; 10: Semi-smooth tongue sole; 11: Atlantic cod; 12: Sablefish; 13: Pacific cod; 14: Pangasius; 15: Chinese sturgeon; 16: Nile tilapia; 17: Atlantic bluefin tuna; 18: Bigeye tuna; 19: Skipjack tuna; 20: Chinese Spanish mackerel; 21: Spanish mackerel; 22: Large yellow croaker; 23: Small yellow croaker; 24: Blank control.

[0019] Figure 2 This is the result of the genomic sensitivity test; Figure 2 The numbers in the table represent: 1-3: 10 ng; 4-6: 1 ng; 7-9: 10 -1ng; 10-12:10 -2 ng; 13-15:10 -3 ng; 16-18: blank control.

[0020] Figure 3 is the mass fraction sensitivity test result; Figure 3 The numbers in the figure represent: 1-4: 100%; 5-8: 10%; 9-12: 1%; 13-16: 0.1%; 17-20: 0.01%; 21-24: blank control.

[0021] Figure 4 The results of the repeatability test. DETAILED DESCRIPTION

[0022] The present invention will be further described with reference to the embodiments.

[0023] Example 1 Specific operation steps: The primer set for rapid identification of pink salmon components of the present invention includes: outer primer F3: 5'-AGATGCGACCTAATACGAAAT-3' (SEQ ID No. 1); outer primer B3: 5'-CAATAAGCCAAACACGTGAT-3' (SEQ ID No. 2); inner primer FIP: 5'-CGGTTTAATGTAGTAAGAACCGACCCCCTGACCATCAATAAGAGT-3' (SEQ ID No. 3); inner primer BIP: 5'-CTGATGCTGAAAGTTGGTGGGTTGTCTGCAACTGATTAATTGC-3' (SEQ ID No. 4); loop primer LB: 5'-CCCGTGTTAGCTGGAGGTTTG-3' (SEQ ID No. 5).

[0024] The kit for rapidly identifying pink salmon components of the present invention comprises the above-mentioned primer set. Further, the kit also comprises 23 μL of LAMP amplification reaction solution, comprising the following components: LAMP premix (2×): 12.5 μL, 0.1 μL each of SEQ ID No. 1 (100 μmol / L) and SEQ ID No. 2 (100 μmol / L), 0.4 μL each of SEQ ID No. 3 (100 μmol / L) and SEQ ID No. 4 (100 μmol / L), 0.2 μL of SEQ ID No. 5 (100 μmol / L), supplemented with ddH2O to 23 μL.

[0025] The method for identifying pink salmon components of the present invention comprises the following steps.

[0026] (1) Extract sample DNA.

[0027] (2) Add 2 μL of the extracted sample DNA to the LAMP amplification reaction solution of the kit.

[0028] (3) In a constant temperature device: 61°C; 60 min.

[0029] The kit also includes positive control, negative control and blank control. Positive control: 2 μL of pink salmon genomic extract is used as a template and added to the LAMP amplification reaction solution (23 μL) of the kit; negative control: 2 μL of genomic extract of other species without positive DNA components is used as a template; blank control: the same volume of ddH2O is used as a template.

[0030] The quality control standard for judging the amplification results is: if any of the following conditions is not met, the experiment is considered invalid: (a) blank control: the reaction solution color is red; (b) negative control: the reaction solution color is red; (c) positive control: the reaction solution color is yellow.

[0031] Result determination: When the reaction solution is yellow, the sample is determined to be positive. When the reaction solution is red, the sample is determined to be negative.

[0032] In the LAMP detection method described above, the sample DNA can be extracted using a commercially available DNA extraction kit, or using the CTAB method, alkaline lysis method, etc.

[0033] Example 2 Specificity Test: Approximately 10 mg (9.90-10.10 mg) of muscle tissue from king salmon, coho salmon, pink salmon, sockeye salmon, dog salmon, Atlantic salmon, rainbow trout, Atlantic mackerel, Japanese mackerel, semi-smooth tongue sole, Atlantic cod, sablefish, Pacific cod, pangasius, Chinese sturgeon, Nile tilapia, Atlantic bluefin tuna, bigeye tuna, bonito, Chinese Spanish mackerel, blue-spotted Spanish mackerel, large yellow croaker, and small yellow croaker was taken, and genomic DNA was extracted and dissolved in 50 μL of buffer. The DNA concentration of the above sample was determined using an ultramicro spectrophotometer, and the DNA concentration was diluted to 5 ng / μL. 2 μL of the extracted sample DNA was added to the LAMP amplification reaction solution of the kit, and pure water was used as the template as a blank control. The reaction solution was placed in a constant temperature device at 61°C for 60 min for isothermal amplification to verify the specificity of the primers.

[0034] Specificity test results such as Figure 1 As shown, the pink salmon genomic DNA reaction solution is yellow, and the other samples are red, indicating that the above primers have good specificity.

[0035] Example 3 Genomic sensitivity test: The concentration of pink salmon genomic DNA was determined using an ultra-micro spectrophotometer. The DNA was diluted 10-fold with ultrapure water. The genomic DNA template concentration was selected to be 5 ng / μL, 5×10 -1 ng / μL, 5×10 -2 ng / μL, 5×10 -3 ng / μL, 5×10 -4 There are 5 dilution levels, 2 μL of genomic DNA from each of the 5 dilution levels was added to the LAMP amplification reaction solution of the kit. The template DNA amounts were 10 ng, 1 ng, 10 ng / μL, and 10 ng / μL, respectively. -1 ng, 10 -2 ng, 10 -3 ng, pure water was used as template and blank control, three parallel replicates were performed for each dilution level, and the reaction solution was placed in a constant temperature device at 61 °C for 60 min for isothermal amplification.

[0036] The results of the sensitivity test of pink salmon genome are as follows Figure 2 As shown, when the amount of genomic DNA ≥ 1 ng, each gradient reaction solution is yellow, and the rest of the gradient reaction solutions are red; this indicates that the sensitivity of genomic DNA can reach 1 ng.

[0037] Example 4 Mass fraction sensitivity test: Pink salmon and tilapia samples were taken, placed in an electric constant temperature blast drying oven, dried at 105°C, and ground into dry powder in a blender. The pink salmon and tilapia dry powders were mixed in different proportions according to dry weight, and then placed in a blender and thoroughly ground and mixed to prepare dry powders with pink salmon mass fractions of 100%, 10%, 1%, 0.1%, and 0.01%, respectively. About 10 mg (9.90-10.10 mg) of dry powder sample was taken, genomic DNA was extracted, and dissolved in 50 μL of buffer. 2 μL of the extracted sample DNA with different mass fractions was added to the LAMP amplification reaction solution of the kit, and pure water was used as a template as a blank control. Four parallel replicates were performed for each concentration, and the reaction solution was placed in a constant temperature device at 61°C for 60 min for isothermal amplification.

[0038] The results of the sensitivity test of pink salmon mass fraction are as follows Figure 3 As shown in the figure, when the mass fraction is ≥1%, the reaction solution at each concentration is yellow, and red reaction solution exists at other concentrations, indicating that the mass fraction sensitivity is 1%.

[0039] Example 5 Repeatability test: About 10 mg (9.90-10.10 mg) of a 1% pink salmon dry powder sample was taken, genomic DNA was extracted, and dissolved in 50 μL of buffer. 2 μL of the extracted sample DNA was added to the LAMP amplification reaction solution of the kit. Eight parallel experiments were performed, and the reaction solution was placed in a constant temperature device at 61°C for 60 min for isothermal amplification.

[0040] Test results such as Figure 4 As shown, the reaction solutions of the eight parallel tests were all yellow, indicating that the method had good repeatability.

Claims

1. A primer set for rapid identification of pink salmon components, characterized in that: It includes a pair of outer primers SEQ ID No.1 and SEQ ID No.2, a pair of inner primers SEQ ID No.3 and SEQ ID No.4 and a loop primer SEQ ID No.

5.

2. A kit for rapid identification of pink salmon components, characterized in that: The kit comprises the primer set according to claim 1 and a LAMP amplification reaction solution.

3. The LAMP amplification reaction solution according to claim 2, characterized in that The total volume is 23 μL, containing LAMP premix (2×): 12.5 μL, 0.1 μL each of SEQ ID No.1 (100 μmol / L) and SEQ ID No.2 (100 μmol / L), 0.4 μL each of SEQ ID No.3 (100 μmol / L) and SEQ ID No.4 (100 μmol / L), 0.2 μL SEQ ID No.5 (100 μmol / L), and ddH2O 9.3 μL.

4. A method for rapid identification of pink salmon components, characterized in that: The specific steps of the method are: (1) Extract sample DNA; (2) Add 2 μL of the extracted sample DNA to the LAMP amplification reaction solution of the kit; (3) In a constant temperature device: 61°C; 60 min; (4) After the reaction is completed, the color change of the reaction liquid is used to determine whether pink salmon components are detected. When the reaction liquid is yellow, the sample is determined to be positive; when the reaction liquid is red, the sample is determined to be negative.

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