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

Abstract

The invention discloses a primer group, a kit and a method for rapidly identifying components of salmon red, belongs to the technical field of nucleic acid detection of marine fish meat products, and particularly relates to a method for rapidly identifying the components of salmon red by using an LAMP (loop-mediated isothermal amplification) technology. The invention provides a primer group for rapid nucleic acid detection and a matched kit, the primer group is composed of an outer primer pair, an inner primer pair and a single loop primer, and the kit comprises the primer group and an optimized LAMP reaction mixed solution. The detection method has the following remarkable characteristics: the DNA of a sample to be detected only needs to be added into the pre-prepared LAMP reaction mixed solution, the detection can be completed by using common constant-temperature equipment, the whole process is about 60 minutes, the color development result can be observed by naked eyes to realize interpretation, if the reaction mixed solution is yellow, the reaction mixed solution is positive, and if the reaction mixed solution is red, the reaction mixed solution is negative. According to the closed tube detection design, the aerosol pollution risk caused by uncovering operation is avoided. The method is excellent in specificity, detection sensitivity (the lowest 10 <-1 > ng genome DNA can be detected or the mass fraction is 1%) and result reproducibility, has the characteristics of simplicity and convenience in operation and low cost, is particularly suitable for resource limited scenes such as grassroots units and field detection, and can remarkably reduce the supervision cost and improve the detection efficiency.

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 components of sockeye salmon using LAMP technology, a specific nucleotide primer set used in the method, and a kit containing the primer set. Background Art

[0002] Salmon, a globally important, high-end commercial aquatic 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 rather refers to a group of salmonids that thrive in the ocean and migrate upstream. These species primarily include Atlantic salmon (Salmo salar), Chinook salmon (Oncorhynchus tshawytscha), Coho salmon (Oncorhynchus kisutch), Scalloped salmon (Oncorhynchus gorbuscha), Sockeye salmon (Oncorhynchus nerka), and King salmon (Oncorhynchus keta). While these species share similar muscle texture and external morphological characteristics, their market value differs significantly. This has led some traders to misrepresent lower-priced species (such as common salmon or rainbow trout) as higher-value Chinook salmon, seriously infringing on consumers' right to know and fair trade.

[0003] Currently, fish ingredient identification relies primarily on real-time fluorescence quantitative PCR (qPCR). While this method offers high sensitivity and specificity, it suffers from significant technical limitations: It relies on a precise temperature control system: the PCR reaction requires precise temperature ramping cycles (denaturation, annealing, and extension), which demands extremely high instrument temperature control accuracy; high equipment costs: the complex and expensive structure of the qPCR instrument increases laboratory hardware investment; and expensive probe reagents: the use of fluorescently labeled probes during the detection process further increases experimental costs. These factors limit the widespread application of this technology in grassroots testing institutions and for rapid on-site screening.

[0004] The loop-mediated isothermal amplification (LAMP) technology used in this invention is an innovative method for nucleic acid molecular detection that has significant advantages over traditional PCR and real-time fluorescence PCR technologies. This technology breaks through the dependence of conventional molecular detection methods on temperature cycling systems, precision instruments, and high-cost reagents, and can achieve efficient and rapid target sequence amplification under constant temperature conditions. Based on LAMP technology, this invention has established a rapid detection system for sockeye salmon ingredients with the following outstanding features: excellent detection specificity and a sensitivity of 10 -1The system is simple to use, with the entire testing process taking only 60 minutes. It utilizes a closed-tube testing method to effectively prevent aerosol contamination. Test results can be directly interpreted visually, eliminating the need for complex instrumentation. The entire experiment requires only a standard constant temperature device. This testing system demonstrates broad application prospects in salmon species identification and fish product composition analysis, making it particularly suitable for use by grassroots testing agencies and market regulators. Its simplicity, speed, and cost-effectiveness provide a highly effective technical solution for aquatic product authenticity verification. Summary of the Invention

[0005] To address these practical issues, the present invention designed a specific LAMP primer set based on the molecular genetic characteristics of sockeye salmon by analyzing the ATP6 gene region. This technical solution utilizes the principle of loop-mediated isothermal amplification to establish an efficient and specific molecular identification method for sockeye salmon components.

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

[0007] The primer set for rapidly identifying components of sockeye salmon comprises: an outer primer F3: 5'-CACTAAATTTAGGCGGTCAC-3' (SEQ ID No. 1); an outer primer B3: 5'-GGTTGATTTCGTATGCCGAT-3' (SEQ ID No. 2); an inner primer FIP: 5'-GAAGTAGGCCCAGTATATTTAGGGTAAATGAGCAGCTCTATTAACCT-3' (SEQ ID No. 3); an inner primer BIP: 5'-ACATTTACCCCTACTACACAACTCTAATCACCGTAGCAAGTCA-3' (SEQ ID No. 4); and a loop primer LB: 5'-CCTAAACATGGGTCTCGCAGT-3' (SEQ ID No. 5).

[0008] The kit for rapidly identifying components of sockeye salmon of the present invention comprises the above-mentioned primer set. Further, the kit also comprises 23 μL of a LAMP reaction mixture, comprising the following components: 12.5 μL of a loop-mediated isothermal amplification reaction premix (2×), 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), and 0.2 μL of SEQ ID No. 5 (100 μmol / L), supplemented with ultrapure water to 23 μL.

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

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

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

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

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

[0014] The validity criteria of the experimental results are as follows (if any condition is not met, the experiment is considered invalid): 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 results were interpreted as follows: After the reaction, the color of the LAMP reaction mixture was used to determine whether red salmon components were detected. If the LAMP reaction mixture was yellow, red indicated that red salmon components were not detected. This method, which uses intuitive color differences to determine results, offers strong visualization and ease of use.

[0016] In this detection method, a variety of mature technical solutions can be used to extract sample DNA: commercially available standardized DNA extraction kits can be used, or conventional laboratory extraction methods can be used, including but not limited to CTAB (cetyltrimethylammonium bromide) extraction method, alkaline lysis extraction method, etc.

[0017] The technical advantages of the present invention are mainly reflected in the following aspects: (1) convenient operation and pollution prevention: the entire detection process takes only 60 minutes, and a closed-tube colorimetric detection mode is adopted. After the reaction, there is no need to open the lid, which avoids the traditional electrophoresis, sequencing and other subsequent processing steps. The results can be interpreted 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 precise temperature control equipment and fluorescence detection systems, and can complete the detection with an ordinary constant temperature device, and no expensive fluorescent labeling probes are required, which greatly reduces the detection cost and is particularly suitable for grassroots units and on-site rapid screening; (3) high specificity: the designed five-fold primer system can accurately identify the target sequence, and the strict matching requirements between the primers and the template ensure the detection specificity, and there will be no cross-reaction between closely related species; (4) excellent sensitivity: the detection limit for genomic DNA can reach 10 -1 The minimum detection limit in mixed samples is 1% by mass, which meets the actual detection needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the specific test result; Figure 1 The numbers in the chart represent: 1: Chinook salmon; 2: Coho salmon; 3: White salmon; 4: Sockeye salmon; 5: 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: Blue-spotted Spanish mackerel; 22: Large yellow croaker; 23: Small yellow croaker; 24: Blank control.

[0019] Figure 2 is the result of genomic sensitivity test; Figure 2 The numbers in the table represent: 1-3: 10 ng; 4-6: 1 ng; 7-9: 10 -1 ng; 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 sockeye salmon components of the present 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); loop primer LB: 5'-CCTAAACATGGGTCTCGCAGT-3' (SEQ ID No. 5).

[0024] The kit for rapidly identifying components of sockeye salmon of the present invention comprises the above-mentioned primer set. Further, the kit also comprises 23 μL of a LAMP reaction mixture, comprising the following components: 12.5 μL of a loop-mediated isothermal amplification reaction premix (2×), 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), and 0.2 μL of SEQ ID No. 5 (100 μmol / L), supplemented with ultrapure water to 23 μL.

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

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

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

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

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

[0030] The validity criteria of the experimental results are as follows (if any condition is not met, the experiment is considered invalid): 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 results were interpreted as follows: After the reaction, the color of the LAMP reaction mixture was used to determine whether red salmon components were detected. If the LAMP reaction mixture was yellow, red indicated that red salmon components were not detected. This method, which uses intuitive color differences to determine results, offers strong visualization and ease of use.

[0032] In this detection method, a variety of mature technical solutions can be used to extract sample DNA: commercially available standardized DNA extraction kits can be used, or conventional laboratory extraction methods can be used, including but not limited to CTAB (cetyltrimethylammonium bromide) extraction method, alkaline lysis extraction method, etc.

[0033] Example 2 Specificity Test: Chinook salmon, coho salmon, scalloped salmon, sockeye salmon, king salmon, Atlantic salmon, rainbow trout, Atlantic mackerel, Japanese mackerel, tongue sole, Atlantic cod, sablefish, Pacific cod, pangasius, Chinese sturgeon, Nile tilapia, Atlantic bluefin tuna, bigeye tuna, skipjack tuna, Chinese Spanish mackerel, bluespot Spanish mackerel, large yellow croaker, and small yellow croaker were selected as test subjects. 9.90-10.10 mg muscle tissue samples were collected from each sample. DNA was extracted and dissolved in 50 μL of buffer. DNA was quantified using an ultramicrospectrophotometer. The DNA concentration of all samples was adjusted to 5 ng / μL. 2 μL of sample DNA solution was added as template to the LAMP reaction mixture, and a blank control of ultrapure water was also established. The reaction was incubated at 61°C for 60 minutes and the color development was observed.

[0034] The specificity verification results showed that ( Figure 1 ), only the reaction mixture of the sockeye salmon sample appeared yellow, while the reaction mixtures of the other fish samples and the blank control were all red, fully demonstrating that the designed primer set has highly specific recognition ability for sockeye salmon components. This result confirms that this detection method can effectively distinguish sockeye salmon from other closely related species and meets practical detection needs.

[0035] Example 3 Genomic sensitivity test: In this study, the concentration of red salmon genomic DNA was accurately determined by ultra-micro spectrophotometer, and then 10-fold serial dilution was performed with ultrapure water to obtain 5 concentration gradients (5 ng / μL to 5×10 -4ng / μL). Take 2 μL of each gradient DNA solution as template and add it into LAMP reaction mixture, the corresponding final template amount is 10ng to 10 -3 ng. An ultrapure water blank control was also set up, and three replicates were set for each concentration. All reactions were incubated at a constant temperature of 61°C for 60 minutes.

[0036] The results of the genomic sensitivity test showed that ( Figure 2 ), when the amount of template DNA ≥ 10 -1 When the concentration was 100 ng, all the reaction mixtures of the replicate samples turned yellow; while the reaction mixtures of the samples below this concentration and the blank control turned red. The results showed that the sensitivity of this method for the detection of red salmon genomic DNA can reach 10 -1 ng level with good reproducibility.

[0037] Example 4 Mass fraction sensitivity test: Red salmon and tilapia muscle tissues were dried at 105°C to constant weight, and the samples were crushed into powders with uniform particle size using a blender. Gradient mixed samples ranging from 100% to 0.01% were accurately prepared according to mass percentage; 9.90-10.10 mg of the mixed powder were weighed for DNA extraction and dissolved in 50 μL of buffer. 2 μL of the DNA extract was taken as a template and added to the LAMP reaction mixture. At the same time, an ultrapure water blank control was set up. Four replicates were set for each gradient, and all reactions were incubated at a constant temperature of 61°C for 60 minutes.

[0038] The results of the mass fraction sensitivity test showed that ( Figure 3 When the sockeye salmon content in the composite sample was ≥1%, all replicates exhibited a typical positive color reaction, with the reaction mixture turning yellow. Samples with a content below 1% and the blank control all exhibited a red reaction mixture. This demonstrates that this method can achieve a quantitative detection limit of 1% by mass for sockeye salmon components in composite samples and exhibits reliable repeatability.

[0039] Example 5: Repeatability Test: To verify the repeatability of this detection method, a 1% mass fraction sockeye salmon mixed sample was used for repeatability testing. Genomic DNA was extracted from 9.90-10.10 mg of the 1% sockeye salmon dry powder mixture, dissolved in 50 μL of buffer, and 2 μL of the DNA solution was added as a template to 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 ) showed that all eight parallel reactions exhibited consistent positive characteristics, with the reaction mixture being yellow. This result confirmed the excellent repeatability and stability of the method at a 1% mass fraction, meeting the reproducibility requirements of the assay.

Claims

1. A primer set for rapid identification of sockeye 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 sockeye salmon ingredients, characterized in that: The kit comprises the primer set according to claim 1 and a LAMP reaction mixture.

3. The LAMP reaction mixture according to claim 2, 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 100 μmol / L SEQ ID No. 1 primer and SEQ ID No. 2 primer, 0.4 μL each of 100 μmol / L SEQ ID No. 3 primer and SEQ ID No. 4 primer, and 0.2 μL of 100 μmol / L SEQ ID No. 5 primer. The volume was made up to 23 μL with ultrapure water.

4. A method for rapid identification of sockeye salmon ingredients, characterized in that: The specific steps of the method are: (1) Extracting genomic DNA from the sample to be tested; (2) Take 2 μL of extracted sample DNA and add it to the LAMP reaction mixture; (3) Place the reaction tube in a 61°C constant temperature device and incubate for 60 min; (4) After the reaction is completed, the color of the LAMP reaction mixture is used to determine whether red salmon components are detected. If the LAMP reaction mixture is yellow, it is determined that red salmon components are detected in the sample. If the LAMP reaction mixture is red, it is determined that red salmon components are not detected in the sample.

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