A primer set, a kit and a method for rapidly identifying components of salmon salmo salar

Abstract

The application discloses a primer group, a kit and a method for rapidly identifying ingredients of red salmon, and belongs to the technical field of nucleic acid detection of marine fish meat products, and particularly relates to a method for rapidly identifying ingredients of red salmon by using LAMP isothermal amplification technology. The application provides a primer group and a matching kit for rapid nucleic acid detection, the primer group is composed of an outer primer pair, an inner primer pair and a single loop primer, and the kit contains the primer combination and an optimized LAMP reaction mixture. The detection method has the following remarkable features: only the DNA of a sample to be detected needs to be added into the pre-prepared LAMP reaction mixture, and the detection can be completed by using a common constant-temperature device, the whole process can be completed in about 60 minutes, the color development result can be observed by naked eyes to realize judgment, the reaction mixture is yellow, which is judged as positive, and the reaction mixture is red, which is judged as negative. The closed-tube detection design avoids the aerosol pollution risk caused by the opening operation. The method is excellent in specificity, detection sensitivity (the minimum detectable genomic DNA is 10 ‑1 ng or 1% mass fraction) and result reproducibility, and has the characteristics of simple operation and low cost, and is particularly suitable for limited resource scenes such as grass-roots units and on-site detection, can significantly reduce the supervision cost and improve the detection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology for marine fish products, specifically relating to a method for rapid identification of components in sockeye salmon using LAMP technology, a specific nucleotide primer set for the method, and a kit containing the primer set. Background Technology

[0002] Salmon, as a globally important high-end economic seafood product, is widely favored by the market for its unique flavor and rich nutritional value. From a taxonomic perspective, "salmon" is not a single species, but refers to a group of salmonid fish that grow in the ocean and migrate upstream to reproduce. These mainly include Atlantic salmon (Salmo salar), chum salmon (Oncorhynchus tshawytscha), coho salmon (Oncorhynchus kisutch), gorbuscha, sockeye salmon (Oncorhynchus nerka), and keta salmon (Oncorhynchus keta). Although these species share similar muscle texture and external morphological characteristics, their market values ​​differ significantly. This leads some operators to sell lower-priced fish (such as common salmon or rainbow trout) as higher-value species (chum salmon), seriously infringing upon consumers' right to know and right to fair trade.

[0003] Currently, fish component identification mainly relies on real-time quantitative PCR (qPCR) technology. Although this method boasts high sensitivity and specificity, it suffers from significant technical limitations: It depends on a precise temperature control system: PCR reactions require precise temperature cycling (denaturation, annealing, extension), demanding extremely high instrument temperature control accuracy; It is costly to equip: qPCR instruments are complex and expensive, increasing laboratory hardware costs; It requires expensive probes: the use of fluorescently labeled probes during detection further increases experimental costs. These factors limit the widespread application of this technology in grassroots testing institutions and rapid on-site screening.

[0004] This invention employs loop-mediated isothermal amplification (LAMP) technology, an innovative nucleic acid molecular detection method that offers significant advantages over traditional PCR and real-time fluorescence PCR. This technology overcomes the dependence of conventional molecular detection methods on temperature cycling systems, precision instruments, and high-cost reagents, enabling efficient and rapid target sequence amplification under constant temperature conditions. Based on LAMP technology, this invention establishes a rapid detection system for components in sockeye salmon, characterized by excellent detection specificity and a sensitivity reaching 10-1. -1The system is rated ng; the operation is simple, with the entire testing process taking only 60 minutes; it adopts a closed-tube testing mode, effectively avoiding aerosol contamination; the test results can be directly interpreted with the naked eye, requiring no complex instruments or equipment; and only a common constant temperature device is needed to complete all experiments. This testing system shows broad application prospects in the fields of salmon variety identification and fish product component analysis, and is particularly suitable for use by grassroots testing institutions and market supervision sites. Its simplicity, speed, and economy provide an efficient technical solution for identifying the authenticity of aquatic products. Summary of the Invention

[0005] To address the aforementioned practical problems, this invention, based on the molecular genetic characteristics of sockeye salmon, designs a specific LAMP primer set by analyzing its ATP6 gene region. This technical approach utilizes the principle of loop-mediated isothermal amplification to establish a highly efficient and specific molecular identification method for sockeye salmon components.

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

[0007] The primer set for rapid identification of components in sockeye salmon of this invention includes: outer primer F3: 5'-CACTAAATTTAGGCGGTCAC-3' (SEQ ID No. 1); outer primer B3: 5'-GGTTGATTTCGTATGCCGAT-3' (SEQ ID No. 2); inner primer FIP: 5'-GAAGTAGGCCCAGTATATTTAGGGTAAATGAGCAGCTCTATTAACCT-3' (SEQ ID No. 3); inner primer BIP: 5'-ACATTTACCCCTACTACACAACTCTAATCACCGTAGCAAGTCA-3' (SEQ ID No. 4); and loop primer LB: 5'-CCTAAACATGGGTCTCGCAGT-3' (SEQ ID No. 5).

[0008] This invention provides a kit for rapid identification of components in sockeye salmon, comprising the aforementioned primer set. Further, the kit includes 23 μL of LAMP reaction mixture, comprising the following components: loop-mediated isothermal amplification reaction premix (2×): 12.5 μL; SEQ ID No. 1 (100 μmol / L) and SEQ ID No. 2 (100 μmol / L) each: 0.1 μL; SEQ ID No. 3 (100 μmol / L) and SEQ ID No. 4 (100 μmol / L) each: 0.4 μL; SEQ ID No. 5 (100 μmol / L) 0.2 μL; and ultrapure water to a final volume of 23 μL.

[0009] The method for identifying components in sockeye salmon according to the present invention includes the following steps.

[0010] (1) Extract genomic DNA from the sample to be tested.

[0011] (2) Take 2 μL of the extracted sample DNA and add it to the LAMP reaction mixture.

[0012] (3) Place the reaction tube in a 61 ℃ constant temperature device and incubate for 60 min.

[0013] The test kit is equipped with three quality control control systems: positive control: 2 μL of standard sockeye salmon genomic DNA is mixed with 23 μL of LAMP reaction mixture as a template; negative control: 2 μL of genomic DNA from a non-target species (such as Atlantic salmon) is used as a template; blank control: 2 μL of ultrapure water is used instead of DNA template.

[0014] The validity criteria for the experimental results are as follows (the experiment is deemed invalid if any condition is not met): Blank control group: the reaction mixture is red; Negative control group: the reaction mixture is red; Positive control group: the reaction mixture is yellow.

[0015] The interpretation criteria are as follows: After the reaction, the color of the LAMP reaction mixture is used to determine whether sockeye salmon is detected. If the LAMP reaction mixture is yellow, sockeye salmon is detected in the sample; if the LAMP reaction mixture is red, sockeye salmon is not detected in the sample. This interpretation method uses intuitive color differences to determine the results, and is characterized by strong visualization and ease of operation.

[0016] In this detection method, DNA extraction from the sample can be performed using a variety of mature technologies: commercially available standardized DNA extraction kits can be used, as well as conventional laboratory extraction methods, including but not limited to CTAB (hexadecyltrimethylammonium bromide) extraction and alkaline lysis extraction.

[0017] The technical advantages of this invention are mainly reflected in the following aspects: (1) Convenient operation and pollution prevention: The entire detection process takes only 60 minutes. The closed tube colorimetric detection mode is adopted. After the reaction, there is no need to open the lid, avoiding the subsequent processing steps such as traditional electrophoresis and sequencing. The results can be read by directly observing the color with the naked eye, which significantly reduces the risk of aerosol pollution; (2) Economical and practical: It gets rid of the dependence on precision temperature control equipment and fluorescence detection system. The detection can be completed by using ordinary constant temperature device, and there is no need for expensive fluorescent label probes, which greatly reduces the detection cost. It is especially suitable for grassroots units and rapid on-site screening; (3) High specificity: The designed five-fold primer system can accurately identify the target sequence. The strict matching requirements of primers and templates ensure the detection specificity. Closely related species will not produce cross-reactions; (4) Excellent sensitivity: The detection limit for genomic DNA can reach 10. -1 The ng level has a limit of detection of 1% by mass in mixed samples, which meets the actual detection requirements. Attached Figure Description

[0018] Figure 1 This is a result of a specific test; Figure 1 The numbers in the text represent: 1: Large-scaled salmon; 2: Silver salmon; 3: Fine-scaled salmon; 4: Solitaire salmon; 5: Chum salmon; 6: Atlantic salmon; 7: Rainbow trout; 8: Atlantic mackerel; 9: Japanese mackerel; 10: Tongue sole; 11: Atlantic cod; 12: Sable scad; 13: Pacific cod; 14: Basa fish; 15: Chinese sturgeon; 16: Nile tilapia; 17: Atlantic bluefin tuna; 18: Bigeye tuna; 19: Skipjack tuna; 20: Chinese mackerel; 21: Blue-spotted mackerel; 22: Large yellow croaker; 23: Small yellow croaker; 24: Blank control.

[0019] Figure 2 Results of genome sensitivity testing; Figure 2 The numbers in the text represent: 1-3: 10 ng; 4-6: 1 ng; 7-9: 10 ng. -1 ng; 10-12:10 -2 ng; 13-15:10 -3 ng; 16-18: Blank control.

[0020] Figure 3 The results are from the mass fraction sensitivity test. Figure 3 The numbers in the table 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 This is the result of a repeatability test. Detailed Implementation

[0022] The present invention will be further described in conjunction with the embodiments.

[0023] Example 1: Specific operating steps: The primer set used in this invention for rapid identification of components in sockeye salmon includes: outer primer F3: 5'-CACTAAATTTAGGCGGTCAC-3' (SEQ ID No. 1); outer primer B3: 5'-GGTTGATTTCGTATGCCGAT-3' (SEQ ID No. 2); inner primer FIP: 5'-GAAGTAGGCCCAGTATATTTAGGGTAAATGAGCAGCTCTATTAACCT-3' (SEQ ID No. 3); inner primer BIP: 5'-ACATTTACCCCTACTACACAACTCTAATCACCGTAGCAAGTCA-3' (SEQ ID No. 4); and loop primer LB: 5'-CCTAAACATGGGTCTCGCAGT-3' (SEQ ID No. 5).

[0024] This invention provides a kit for rapid identification of components in sockeye salmon, comprising the aforementioned primer set. Further, the kit includes 23 μL of LAMP reaction mixture, comprising the following components: loop-mediated isothermal amplification reaction premix (2×): 12.5 μL; SEQ ID No. 1 (100 μmol / L) and SEQ ID No. 2 (100 μmol / L) each: 0.1 μL; SEQ ID No. 3 (100 μmol / L) and SEQ ID No. 4 (100 μmol / L) each: 0.4 μL; SEQ ID No. 5 (100 μmol / L) 0.2 μL; and ultrapure water to a final volume of 23 μL.

[0025] The method for identifying components in sockeye salmon according to the present invention includes the following steps.

[0026] (1) Extract genomic DNA from the sample to be tested.

[0027] (2) Take 2 μL of the extracted sample DNA and add it to the LAMP reaction mixture.

[0028] (3) Place the reaction tube in a 61 ℃ constant temperature device and incubate for 60 min.

[0029] The test kit is equipped with three quality control control systems: positive control: 2 μL of standard sockeye salmon genomic DNA is mixed with 23 μL of LAMP reaction mixture as a template; negative control: 2 μL of genomic DNA from a non-target species (such as Atlantic salmon) is used as a template; blank control: 2 μL of ultrapure water is used instead of DNA template.

[0030] The validity criteria for the experimental results are as follows (the experiment is deemed invalid if any condition is not met): Blank control group: the reaction mixture is red; Negative control group: the reaction mixture is red; Positive control group: the reaction mixture is yellow.

[0031] The interpretation criteria are as follows: After the reaction, the color of the LAMP reaction mixture is used to determine whether sockeye salmon is detected. If the LAMP reaction mixture is yellow, sockeye salmon is detected in the sample; if the LAMP reaction mixture is red, sockeye salmon is not detected in the sample. This interpretation method uses intuitive color differences to determine the results, and is characterized by strong visualization and ease of operation.

[0032] In this detection method, DNA extraction from the sample can be performed using a variety of mature technologies: commercially available standardized DNA extraction kits can be used, as well as conventional laboratory extraction methods, including but not limited to CTAB (hexadecyltrimethylammonium bromide) extraction and alkaline lysis extraction.

[0033] Example 2 Specificity Test: The following fish species were selected as test subjects: large-scaled salmon, silver salmon, slender-scaled salmon, sockeye salmon, salmon, Atlantic salmon, rainbow trout, Atlantic mackerel, Japanese mackerel, tongue sole, Atlantic cod, sable scad, Pacific cod, basa fish, Chinese sturgeon, Nile tilapia, Atlantic bluefin tuna, bigeye tuna, skipjack tuna, Chinese mackerel, blue-spotted mackerel, large yellow croaker, and small yellow croaker. Muscle tissue samples of each species (9.90-10.10 mg) were taken, and after DNA extraction, the DNA was dissolved in 50 μL of buffer. Quantification was performed using an ultra-micro spectrophotometer, and the DNA concentration of all samples was uniformly adjusted to 5 ng / μL. 2 μL of the sample DNA solution was used as a template and added to the LAMP reaction mixture, with an ultrapure water blank control. The reaction was carried out at a constant temperature of 61℃ for 60 minutes, and the color development was observed.

[0034] Specificity verification results showed that ( Figure 1 Only the reaction mixture of the sockeye salmon sample showed a yellow color, while the reaction mixtures of the other fish samples and the blank control were red, fully demonstrating that the designed primer set has a high specificity for recognizing sockeye salmon components. This result confirms that this detection method can effectively distinguish sockeye salmon from other closely related species, meeting practical detection needs.

[0035] Example 3: Genomic Sensitivity Test: In this study, the concentration of sockeye salmon genomic DNA was accurately determined using an ultra-micro spectrophotometer, followed by 10-fold serial dilutions with ultrapure water to obtain five concentration gradients (5 ng / μL to 5 × 10⁻⁶). -4ng / μL). Take 2 μL of each gradient DNA solution as template and add it to the LAMP reaction mixture, corresponding to final template amounts of 10 ng to 10 ng / μL respectively. -3 ng. An ultrapure water blank control was set up at the same time, and three replicates were set up for each concentration. All reactions were incubated at a constant temperature of 61℃ for 60 minutes.

[0036] The results of the genome sensitivity test showed that ( Figure 2 When the amount of template DNA is ≥10 -1 At a concentration of ng, all replicate sample reaction mixtures turned yellow; while samples at lower concentrations and the blank control reaction mixtures turned red. The results indicate that this method has a sensitivity of up to 10 for detecting sockeye salmon genomic DNA. -1 The ng level is well-reproducible.

[0037] Example 4: Mass fraction sensitivity test: Somalia salmon and tilapia muscle tissues were dried at 105 °C to constant weight. The samples were then pulverized into uniform powder using a mixer. Gradient mixtures of 100% to 0.01% by mass were precisely prepared. 9.90-10.10 mg of the mixed powder were weighed for DNA extraction. The DNA was dissolved in 50 μL of buffer. 2 μL of the DNA extract was used as a template and added to the LAMP reaction mixture. An ultrapure water blank control was also set up. Four replicates were set up for each gradient. All reactions were incubated at 61 °C for 60 minutes.

[0038] The results of the mass fraction sensitivity test showed that ( Figure 3 When the content of sockeye salmon in the mixed sample is ≥1%, all parallel samples show a typical positive colorimetric reaction, i.e., the color of the reaction mixture is yellow; while the sample group with a content of less than 1% and the blank control all show a red reaction mixture. This indicates that the limit of quantitative detection of sockeye salmon components in the composite sample by this method can reach 1% by mass fraction, and it has reliable repeatability.

[0039] Example 5 Repeatability Test: To verify the repeatability of this detection method, a 1% (w / w) mixed sample of sockeye salmon was selected for a repeatability test. 9.90-10.10 mg of the mixed dry powder containing 1% sockeye salmon was weighed, genomic DNA was extracted, dissolved in 50 μL buffer, and 2 μL of the DNA solution was used as a template in the LAMP reaction mixture. Eight independent parallel reactions were set up and incubated at 61°C for 60 minutes.

[0040] Repeatability test results ( Figure 4 The results showed that all eight parallel reactions exhibited a consistent positive reaction characteristic, namely, the reaction mixture was yellow in color. This result confirms that the method has excellent repeatability and stability at a 1% mass fraction, meeting the repeatability requirements for detection.

Claims

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

5.

2. A kit for rapid identification of salmonid species, characterized in that, The kit includes the primer set as described in claim 1 and the LAMP reaction mixture.

3. A LAMP reaction mix, characterized in that The total volume was 23 μL, containing 12.5 μL of 2× loop-mediated isothermal amplification reaction premix, 0.1 μL each of SEQ ID No. 1 and SEQ ID No. 2 primers at 100 μmol / L, 0.4 μL each of SEQ ID No. 3 and SEQ ID No. 4 primers at 100 μmol / L, and 0.2 μL of SEQ ID No. 5 primer at 100 μmol / L, and then made up to 23 μL with ultrapure water.

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