Primer group, kit and method for rapidly and visually detecting components of sea eel

Abstract

The invention discloses a primer group, a kit and a method for rapidly and visually detecting components of sea eel, belongs to the technical field of nucleic acid detection of marine fish meat products, and particularly relates to a method for rapidly and visually detecting components of sea eel by using an LAMP (Loop-Mediated Isothermal Amplification) technology. The invention provides a set of primer combination adaptive to rapid nucleic acid detection and a matched kit, the primer combination is composed of an outer primer pair, an inner primer pair and a loop primer, and the kit integrates the primer combination with an optimized LAMP reaction mixed solution. The detection technology has multiple core advantages that in the detection process, only DNA of a sample to be detected needs to be added into a prefabricated LAMP reaction mixed solution, and detection can be started by using conventional constant-temperature equipment. The whole process only needs 60 minutes, the interpretation can be completed by observing the developing result with naked eyes, the reaction liquid is positive if yellow, and the reaction liquid is negative if red. Due to the design of closed tube detection, the problem of aerosol pollution possibly caused by uncovering operation is fundamentally avoided. The method is strong in specificity and high in detection sensitivity, and can detect 1 ng of genome DNA or a sample with the mass fraction of 1% at least. The method is good in detection result reproducibility, has the characteristics of simplicity and convenience in operation and low cost, and is particularly suitable for basic agricultural fry breeding units with limited funds and resource shortage scenes such as field supervision and detection and rapid port customs clearance. When the method is applied to primary field detection and identification of the sea eel germplasm, the supervision cost can be greatly reduced, and meanwhile, the detection and identification working efficiency is remarkably improved.

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 and visual detection of eel components using LAMP technology, a specific nucleotide primer set for the method, and a kit containing the primer set. Background Technology

[0002] Conger eels (Muraenesox cinereus) belong to the order Anguilliformes, family Murenesocidae, and genus Murenesox. They are warm-water, near-bottom fish, widely distributed in the Red Sea of ​​East Africa, the northern Indian Ocean, and Myanmar and Malaysia in the Northwest Pacific. In my country, their main production area is the East China Sea. Conger eel meat is thick, fine-textured, delicious, and high in fat, possessing a strong gel-forming ability, making it an ideal raw material for producing high-end surimi products. In recent years, adulteration and counterfeiting have been rampant in the surimi market, frequently reported in the news. For example, seafood balls are basically made by mixing surimi with flavorings, containing no actual seafood; imitation seafood products are also mostly mixtures of inferior surimi and flour, causing significant losses to consumers. In recent years, the international market demand for conger eels has been substantial, and conger eel farming has developed rapidly along the coasts of Fujian and Zhejiang. Due to the similar external morphology of various eel species, especially at the fry stage, they are difficult to distinguish. To prevent adulteration of conger eel meat and mixing of different strains, establishing molecular identification methods for conger eels is of great practical significance.

[0003] Currently, the identification of fish components mainly relies on real-time fluorescence PCR technology. While this technology excels in detection sensitivity and specificity, it also has significant technical limitations: First, it is highly dependent on a precise temperature control system. The PCR reaction requires precise temperature cycling (including denaturation, annealing, and extension steps), placing extremely high demands on the instrument's temperature control accuracy. Second, the equipment cost is high; the complex structure of real-time fluorescence PCR instruments leads to substantial expenses, undoubtedly increasing the laboratory's hardware investment. Third, probe reagents are expensive; the use of fluorescently labeled probes during detection further raises the overall experimental cost. These factors combined limit the widespread adoption and application of this technology in grassroots testing institutions and on-site rapid screening scenarios. Microsatellite markers are commonly used molecular markers for germplasm identification, possessing advantages such as co-dominance, high resolution, high repeatability, and high reliability. They are the most widely used nuclear genomic markers in aquatic research. This method typically uses a dozen or so pairs of microsatellite primers combined with software analysis to identify different fish species. These methods use complex primers, are cumbersome to operate, and are time-consuming. They also require specialized molecular biology software and demand a high level of expertise from the testing personnel. They are not suitable for grassroots laboratories and cannot meet the requirements for rapid customs clearance at ports.

[0004] The loop-mediated isothermal amplification (LAMP) technology used in this invention is a novel nucleic acid molecular detection method with significant advantages over traditional PCR, real-time fluorescence PCR, and microsatellite labeling technologies. This technology breaks the dependence of conventional molecular detection methods on temperature cycling systems, precision instruments, and high-cost reagents, enabling efficient and rapid amplification of target sequences under isothermal conditions. Based on LAMP technology, this invention constructs a rapid detection system for eel components, with the following key features: excellent detection specificity, with sensitivity reaching the 1 ng level; simple and convenient operation, requiring only 60 minutes for the entire detection process; closed-tube detection method, effectively preventing aerosol contamination; detection results can be directly observed and judged with the naked eye, without the need for complex instruments; and all experimental operations can be completed using only a common isothermal device. This detection system shows broad application potential in fish identification and fish product component analysis, and is particularly suitable for use in grassroots institutions and market supervision sites. Its simplicity, speed, and economy provide an efficient technical solution for identifying genuine and counterfeit eels. Summary of the Invention

[0005] To address the aforementioned practical problems, this invention, based on the molecular genetic characteristics of moray eels, designs a specific LAMP primer set by analyzing their mitochondrial ND2 gene. This technical solution utilizes the principle of loop-mediated isothermal amplification to establish a highly efficient and specific method for visually detecting moray eel components.

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

[0007] The primer set for rapid and visual detection of components in moray eels according to this invention includes: outer primer F3: 5'-TGTTCATGTTAACATCCACC-3' (SEQ ID No. 1); outer primer B3: 5'-ATAGGCTGAGAAGGGCAC-3' (SEQ ID No. 2); inner primer FIP: 5'-GTCCGGAGAGTGATAGTAATGTCATGTAGCATCAAGCTGGTCAA-3' (SEQ ID No. 3); inner primer BIP: 5'-GACAGGTTTCCTACCAAAAATCCTGTTACTGCTATAATGGCGATT-3' (SEQ ID No. 4); and loop primer LB: 5'-CCCGTGTTAGCTGGAGGTTTG-3' (SEQ ID No. 5).

[0008] The present invention provides a kit for rapid and visual detection of components in moray eels, comprising the aforementioned primer set. Further, the kit includes 24 μL of LAMP reaction mixture, comprising the following components: 12.5 μL of LAMP 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), 0.2 μL of SEQ ID No. 5 (100 μmol / L), and brought to a final volume of 24 μL with ultrapure water.

[0009] The method for visually detecting components in moray eels according to the present invention includes the following steps.

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

[0011] (2) Take 1 μL of the extracted sample DNA and add it to the LAMP reaction mixture.

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

[0013] The test kit is equipped with three quality control control systems: positive control: 1 μL of standard genomic DNA of moray eel is used as a template and mixed with 24 μL of LAMP reaction mixture; negative control: 1 μL of genomic DNA of a non-target species (such as American eel) is used as a template; blank control: 1 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 eel components are detected. If the LAMP reaction mixture is yellow, eel components are detected in the sample; if the LAMP reaction mixture is red, eel components are 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 color development detection mode is adopted. After the reaction is completed, there is no need to open the lid, which saves the traditional electrophoresis, sequencing and other subsequent processing links. The result can be interpreted by directly observing the color change with the naked eye, avoiding aerosol pollution; (2) Economical and practical: It no longer relies on precise temperature control equipment and fluorescence detection system. It can carry out detection using ordinary constant temperature device, and there is no need to use expensive fluorescent label probes, which greatly reduces the cost required for detection. It is very suitable for use in grassroots units and on-site rapid screening scenarios; (3) High specificity: The designed five primer system can accurately identify the target sequence. Since there is a strict matching requirement between the primer and the template, the specificity of the detection is guaranteed. Even closely related species of the same genus will not have cross-reaction; (4) Excellent sensitivity: The detection limit for genomic DNA can reach 1 ng level. In mixed samples, the minimum target component with a mass fraction of 1% can be detected, which can fully meet the needs of actual detection work. Attached Figure Description

[0018] Figure 1 For the reaction temperature and reaction time experiments; Figure 1The numbers in the table represent: 1: 65 ℃; 2: 64.7 ℃; 3: 64.2 ℃; 4: 63.2 ℃; 5: 62 ℃; 6: 61 ℃; 7: 60.3 ℃; 8: 60 ℃.

[0019] Figure 2 This is a result of a specific test; Figure 2 The labels in the text represent: 1: moray eel; 2: cloning vector pUC57 carrying the brown moray eel ND2 gene; 3: Japanese eel; 4: American eel; 5: conger eel; 6: spotted eel; 7: rainbow trout; 8: Atlantic mackerel; 9: Japanese mackerel; 10: tongue sole; 11: Atlantic cod; 12: sable smelt; 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.

[0020] Figure 3 This is a comparison diagram of the sequencing results of the amplified products.

[0021] Figure 4 Results of genome sensitivity testing; Figure 4 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: Blank control.

[0022] Figure 5 The results are from the mass fraction sensitivity test. Figure 5 The numbers in the table represent: 1-3: 100%; 4-6: 10%; 7-9: 1%; 10-12: 0.1%; 13-15: blank control.

[0023] Figure 6 This is the result of a repeatability test. Detailed Implementation

[0024] The present invention will be further described in conjunction with the embodiments.

[0025] Example 1: Reaction temperature and time experiment: Using 10 ng / μL eel genomic DNA, 1 μL of sample DNA solution was added to the LAMP reaction mixture as a template. Eight independent parallel reactions were set up and placed in a PCR instrument. Temperature gradients of (65, 64.7, 64.2, 63.2, 62, 61, 60.3, 60) °C were set, and the color development was observed at 30 min, 40 min, 50 min, and 60 min.

[0026] The results are as follows Figure 1 At 30 min, 40 min, and 50 min, all reactions were red. At 60 min, a yellow color appeared, and the color was the yellowest at 60 °C. Therefore, incubation at 60 °C for 60 min was selected as the reaction condition.

[0027] Example 2 Specificity Test: Genomic DNA from several species, including moray eel, Japanese eel, American eel, conger eel, spotted eel, 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, along with the cloning vector pUC57 carrying the brown moray eel ND2 gene, was used as templates. The DNA concentration of these samples was determined using an ultra-micro spectrophotometer. The concentration of moray eel genomic DNA was adjusted to 10 ng / μL, and the concentration of the DNA from the remaining samples was uniformly adjusted to 100 ng / μL. 1 μL of the sample DNA solution was added as a template to the LAMP reaction mixture, with an ultrapure water blank control. The reaction was carried out at 60 ℃ for 60 minutes, and the color development was observed. The amplified products were sequenced, and the sequencing results were compared using NCBI.

[0028] Specificity verification results showed that ( Figure 2 Only the reaction mixture of the moray eel sample was yellow, while the reaction mixtures of the other fish samples and the blank control were red. The sequencing sequences of the positive sample amplification products were BLAST-aligned using NCBI, and the sample sequences showed 100% similarity to the moray eel mitochondrial gene sequence. Figure 3 As shown, the designed primer set fully demonstrates its high specificity in recognizing components of moray eels. This result confirms that the proposed detection method can effectively distinguish moray eels from other closely related species, meeting practical detection needs.

[0029] Example 3: Genomic Sensitivity Assay: In this study, the concentration of eel genomic DNA was accurately determined using an ultra-micro spectrophotometer, followed by 10-fold serial dilutions with ultrapure water to obtain four concentration gradients (10 ng / μL to 10 ng / μL). -2 (ng / μL). Take 1 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. -2 ng. An ultrapure water blank control was set up, and three replicates were set up for each concentration. All reactions were incubated at a constant temperature of 60 ℃ for 60 minutes.

[0030] The results of the genome sensitivity test showed that ( Figure 4When the amount of template DNA was ≥1 ng, all replicate sample reaction mixtures were yellow; while samples with concentrations below this and the blank control showed red reaction mixtures. The results indicate that this method has a sensitivity of up to 1 ng for detecting eel genomic DNA and exhibits good reproducibility.

[0031] Example 4: Mass fraction sensitivity test: The muscle tissue of eel and basa fish was dried at 105 °C to constant weight. The samples were pulverized into powder with uniform particle size using a mixer. Gradient mixed samples from 100% to 0.1% by mass percentage were precisely prepared. 20 mg of the mixed powder was weighed for DNA extraction and dissolved in 50 μL of buffer. 1 μL of the DNA extract was used as a template and added to the LAMP reaction mixture. An ultrapure water blank control was set up at the same time. Three replicate experiments were set up for each gradient. All reactions were incubated at 60 °C for 60 minutes.

[0032] The results of the mass fraction sensitivity test showed that ( Figure 5 When the eel content in the mixed sample was ≥1%, all parallel samples showed a typical positive colorimetric reaction, i.e., the reaction mixture was yellow; while the sample group with a content of less than 1% and the blank control showed a red reaction mixture. This indicates that the limit of quantitative detection of eel components in the composite sample by this method can reach 1% by mass fraction, and it has reliable repeatability.

[0033] Example 5 Repeatability Test: To verify the repeatability of this detection method, a 1% (w / w) mixed sample of conger eel was selected for a repeatability test. 20 mg of mixed dry powder containing 1% conger eel was weighed, genomic DNA was extracted, dissolved in 50 μL buffer, and 1 μL of the DNA solution was used as a template and added to the LAMP reaction mixture. Eight independent parallel reactions were set up and incubated at 60°C for 60 minutes.

[0034] Repeatability test results ( Figure 6 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 and visual detection of components in moray eels, 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 rapid and visual reagent kit for detecting components in moray eels, characterized in that, The kit includes the primer set as described in claim 1 and the LAMP reaction mixture.

3. The LAMP reaction mixture according to claim 2, characterized in that, The total volume is 24 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 of SEQ ID No.5 (100 μmol / L), and bring the volume to 24 μL with ultrapure water.

4. A method for rapid and visual detection of components in moray eels, characterized in that, The specific steps of the method are as follows: (1) Extract genomic DNA from the sample to be tested; (2) Take 1 μL of the extracted sample DNA and add it to the LAMP reaction mixture; (3) Place the reaction tube in a 60 ℃ 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 the eel component is detected. If the LAMP reaction mixture is yellow, the eel component is detected in the sample. If the LAMP reaction mixture is red, the eel component is not detected in the sample.

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