Primer group for identifying meat sources of beef and mutton products as well as use method and application of primer group

Abstract

The invention discloses a primer group for identifying meat sources of beef and mutton products as well as a use method and application thereof, and belongs to the technical field of molecular biology. In order to solve the problem that in the prior art, specificity, universality, sensitivity and low cost are not guaranteed, and meanwhile synchronous and rapid detection of meat sources of beef and mutton products is achieved, the primer group for identifying the meat sources of the beef and mutton products is provided, and a multiple PCR detection method which is high in specificity, high in sensitivity, synchronous in multiple targets and easy and convenient to operate is achieved. A complete technical scheme is provided for rapid, accurate and generalizable identification of beef and mutton products, and the efficiency and reliability of food safety supervision and market monitoring are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology, and particularly relates to a primer set for identifying the meat source of beef and mutton products and a use method and application thereof. BACKGROUND

[0002] With the improvement of living standards and the rapid development of the food industry, beef and mutton and their products have a sustained growth in consumption due to their high protein, low fat and unique nutritional value. However, the long growth cycle and low feed conversion efficiency of beef and mutton result in a unit price that has been maintained at 2-4 times that of pork and 3-6 times that of duck meat for a long time. The huge price difference makes beef and mutton one of the highest adulteration rates of meat in the world. Driven by economic benefits, the adulteration of meat products is becoming increasingly complex. In addition to the common use of low-priced meat (such as pork, duck and chicken) to pretend, it is also found that special economic animal fur (such as mink, raccoon and fox) and even rat meat are added. Therefore, it is of great significance to develop a variety of conventional and unconventional animal-derived component detection methods for identifying the meat source of beef and mutton products to ensure food safety, regulate market order and crack down on illegal and irregular behavior.

[0003] In the prior art, there are certain limitations in the traditional identification methods of meat products. (1) Sensory evaluation and physicochemical indicators: relying on color, odor, texture and hydroxyproline / inosinic acid ratio, etc. Physicochemical parameters, the identification accuracy is ≤60% in the deep processing scene of meat paste, high-temperature sterilization, pickling and seasoning, and cannot distinguish between closely related species; (2) Immunological methods: based on protein antigen-antibody reaction, which has the advantage of simple operation. However, after high-temperature sterilization or long-term freezing of meat, protein denaturation, cross-linking and hydrolysis occur, leading to loss of antibody recognition epitopes, with a false negative rate as high as 25%. At the same time, the cross-reaction of antibodies with homologous proteins (such as hemoglobin and troponin) also easily leads to false positives; (3) Conventional DNA detection methods: compared with the above methods, DNA-based detection technology has stronger stability and higher species specificity, which can effectively break through the limitations of traditional methods and has become the mainstream technology route for meat source identification. However, conventional methods such as single PCR can only detect one species at a time, and the detection cycle is long and the efficiency is low when facing "multi-species mixing". Although the sensitivity of fluorescent quantitative PCR can reach 0.1%, it requires real-time fluorescence equipment, standard curve establishment and professional operation, and the detection cost is ≥180 yuan / sample, which is difficult to promote in county-level laboratories, port sites or enterprise raw material acceptance links, etc. In summary, developing a DNA technology solution that combines high specificity and sensitivity with high efficiency, low cost, and simultaneous detection of multiple targets is crucial. Multiplex PCR technology can effectively overcome the shortcomings of the methods mentioned above. It can simultaneously amplify multiple species-specific target sequences in the same reaction system, providing an ideal solution for the efficient simultaneous detection of cattle, sheep, and common adulterated meats. However, designing multiplex PCR primers for multi-species identification requires simultaneously meeting the dual requirements of stable amplification in the target species and no amplification in any non-target species. Achieving this goal faces multiple technical obstacles, including the risk of cross-amplification due to the high similarity of sequences in closely related species, the lack of universality caused by genetic polymorphism within species, and the complexity of balancing primer interactions and amplification efficiency in the multiplex PCR system.

[0004] To overcome the aforementioned technical bottlenecks, this invention aims to construct a high-efficiency, low-cost multiplex PCR detection system that does not rely on a quantitative fluorescence platform and only requires a conventional PCR instrument and agarose gel electrophoresis. This system can simultaneously identify bovine, ovine, and various conventional and unconventional animal-derived components in a single reaction, filling a technical gap in the current food safety regulatory chain and providing a powerful on-site monitoring tool for grassroots testing units, ports, and enterprises. Summary of the Invention

[0005] This invention addresses the lack of existing technologies that can simultaneously, rapidly, and on-site detect "common adulteration (pig, duck, chicken) + covert adulteration (mink, raccoon dog, fox, rat)" in beef and mutton products while ensuring specificity, universality, sensitivity, and low cost. It provides a primer set and multiplex PCR detection system and method for identifying the meat source of beef and mutton products.

[0006] One of the objectives of this invention is to provide a primer set for identifying the meat source of beef and mutton products, the primer set including specific primer pairs for cattle, sheep, pigs, chickens, ducks, foxes, raccoons, minks, and mice; The upstream primer sequence for the bovine sample is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2. The upstream primer sequence for the sheep is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4. The upstream primer sequence for the pig is shown in SEQ ID NO.5, and the downstream primer sequence is shown in SEQ ID NO.6. The upstream primer sequence for the chicken is shown in SEQ ID NO.7, and the downstream primer sequence is shown in SEQ ID NO.8. The upstream primer sequence for the duck is shown in SEQ ID NO.9, and the downstream primer sequence is shown in SEQ ID NO.10. The upstream primer sequence for the fox is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQ ID NO.12. The upstream primer sequence for the raccoon dog is shown in SEQ ID NO.13, and the downstream primer sequence is shown in SEQ ID NO.14. The upstream primer sequence for the mink is shown in SEQ ID NO.15, and the downstream primer sequence is shown in SEQ ID NO.16. The upstream primer sequence for the mouse is shown in SEQ ID NO.17, and the downstream primer sequence is shown in SEQ ID NO.18.

[0007] The second objective of this invention is to provide a kit for identifying the meat source of beef and mutton products, the kit containing the aforementioned primer set.

[0008] The third objective of this invention is to provide the application of the above-mentioned primer set and kit in the detection of meat sources in beef and mutton products.

[0009] The fourth objective of this invention is to provide a method for identifying the meat source of beef and mutton products, comprising the following steps: S1: Extract genomic DNA from the sample to be tested; S2: Using the DNA extracted in S1 as a template, multiplex PCR amplification was performed using the above primer set to obtain the amplification product; S3: Perform electrophoretic detection on the amplification products obtained in S2, and identify the meat source based on the detection results.

[0010] In a preferred embodiment of the present invention, the multiplex PCR amplification in S2 includes three amplification systems: the first system consists of pigs, mice, and ducks; the second system consists of cattle, raccoon dogs, and chickens; and the third system consists of foxes, minks, and sheep.

[0011] In a preferred embodiment of the present invention, the multiplex PCR amplification system in S2 includes the primer set described above; the thermostable DNA polymerase is a hot-start Taq DNA polymerase, used at a concentration of 0.02-0.1 U / μL; the deoxyribonucleoside triphosphate (dNTPs) are a mixture of dATP, dTTP, dCTP, and dGTP, with each dNTP in the mixture having a final concentration of 100-400 μM; Mg 2+ The ions were provided by magnesium salts at a final concentration of 1.5–3.0 mM; the reaction buffer was Mg-free. 2+ The 10×PCR buffer should be diluted to a working concentration before use.

[0012] In a preferred embodiment of the present invention, the concentrations of the primer sets in the multiplex PCR detection system are as follows: the final concentration of the specific primer pairs for cattle, pigs, chickens, ducks, foxes, minks, and mice in the primer set is 0.2 μM; the final concentration of the specific primer pairs for sheep and raccoon dogs in the primer set is 0.4 μM.

[0013] In a preferred embodiment of the present invention, the conditions for multiplex PCR amplification in S2 are: pre-denaturation at 95℃ for 3-10 min; denaturation at 95℃ for 20-45 s, annealing at 56-60℃ for 10-60 s, extension at 72℃ for 20-45 s, for 25-45 cycles; and extension at 72℃ for 3-10 min.

[0014] In a preferred embodiment of the present invention, the electrophoretic detection in S3 is agarose gel electrophoresis or capillary electrophoresis, and the meat source is determined according to the length of the amplified fragment.

[0015] In a preferred embodiment of the present invention, the criterion for determining the meat source is: If the amplified fragment length is 132 bp, then the meat source of the sample to be tested is cattle; If the amplified fragment length is 250 bp, then the meat source of the sample to be tested is sheep; If the amplified fragment length is 120 bp, then the meat source of the sample to be tested is pig; If the length of the amplified fragment is 193 bp, then the meat source of the sample to be tested is chicken; If the amplified fragment length is 187 bp, then the meat source of the sample to be tested is duck; If the amplified fragment length is 142 bp, then the meat source of the sample to be tested is fox. If the amplified fragment length is 154 bp, then the meat source of the sample to be tested is raccoon dog; If the amplified fragment length is 179 bp, then the meat source of the sample to be tested is mink; If the amplified fragment length is 153 bp, then the meat source of the sample to be tested is mouse.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides a primer set for identifying the meat source of beef and mutton products. The primer design takes into account both similar sequences of closely related species and intraspecific polymorphism, and selects primer combinations with high specificity and stability, which can achieve stable amplification in the target species and avoid cross-reaction. At the same time, this invention constructs a multiplex PCR detection system, which, through scientific grouping and concentration optimization, successfully achieves simultaneous identification of adulterated animals such as cattle, sheep, pigs, chickens, ducks, dogs, foxes, raccoons, and rats.

[0017] The primer set and multiplex PCR detection system for identifying the meat source of beef and mutton products provided by this invention have the following significant advantages compared with existing technologies: (1) High specificity: Each primer pair has high amplification efficiency and strong stability in the target species, and no non-specific amplification in non-target species. It can accurately distinguish closely related species and effectively avoid cross-reaction. (2) High-throughput parallel detection: It can detect up to 9 species components simultaneously, which is especially suitable for samples with complex composition and may contain multiple adulterants or substitutes, thus improving detection efficiency and throughput; (3) Strong multi-target compatibility: The detection targets are distributed to three independent multiplex PCR systems. The primers in each system are synergistically optimized and have good compatibility; and the reaction conditions of the three systems are completely consistent, which can be run synchronously on the same PCR instrument, realizing a significant increase in detection throughput and a great simplification of operation; (4) High sensitivity: Reliable detection can still be achieved under low doping ratio conditions (target species DNA template concentration as low as 0.1 ng / μL), ensuring the accuracy of detection results; (5) Simple operation and low cost: No expensive fluorescence quantitative PCR equipment is required. Multiple species identification can be completed by relying solely on conventional gel electrophoresis, which reduces experimental costs and facilitates the promotion of testing in grassroots laboratories and on-site testing. (6) Wide applicability: It can cover beef and mutton products with different processing techniques such as fresh, frozen, pickled, and boiled. It has strong anti-interference ability, stable and reliable results, and is suitable for the detection of different types of samples. (7) Potential for reagent kit development: By integrating optimized primer combinations, PCR premix, positive control and standardized operating procedures into a ready-to-use test kit, detection efficiency, consistency and reproducibility can be significantly improved, providing a fast, reliable and easy-to-promote complete technical solution for food traceability, market supervision and enterprise quality control.

[0018] Overall, this invention, through innovations in primer design and system optimization, achieves a highly specific, highly sensitive, multi-target simultaneous, easy-to-operate, and widely applicable multiplex PCR detection method. It provides a complete technical solution for the rapid, accurate, and widely applicable identification of beef and mutton products, significantly improving the efficiency and reliability of food safety supervision and market monitoring.

[0019] This invention provides a method for identifying the meat source of beef and mutton products. Unlike methods that rely on expensive quantitative PCR, this system optimizes PCR amplification conditions so that the amplified products are clearly distinguishable in terms of fragment length or band pattern. Detection can be completed using only conventional gel electrophoresis. It has the advantages of being simple, low-cost, and easy to promote, making it suitable for widespread application in grassroots laboratories and food safety supervision. Attached Figure Description

[0020] Figure 1The image shows the PCR amplification results of pig-specific primers in DNA templates from pigs, mice, raccoons, chickens, ducks, sheep, foxes, minks, and cattle. Figure 2 The image shows the PCR amplification results of mouse-specific primers in DNA templates from pigs, mice, raccoons, chickens, ducks, sheep, foxes, minks, and cattle. Figure 3 The results of PCR amplification of DNA templates from pigs, mice, raccoons, chickens, ducks, sheep, foxes, minks, and cattle using raccoon dog-specific primers. Figure 4 The image shows the PCR amplification results of chicken-specific primers in DNA templates from pigs, mice, raccoons, chickens, ducks, sheep, foxes, minks, and cattle. Figure 5 The image shows the PCR amplification results of duck-specific primers in DNA templates from pigs, mice, raccoons, chickens, ducks, sheep, foxes, minks, and cattle. Figure 6 The image shows the PCR amplification results of sheep-specific primers in DNA templates from pigs, mice, raccoons, chickens, ducks, sheep, foxes, minks, and cattle. Figure 7 The image shows the PCR amplification results of fox-specific primers in DNA templates from pigs, mice, raccoons, chickens, ducks, sheep, foxes, minks, and cattle. Figure 8 The image shows the PCR amplification results of mink-specific primers in DNA templates from pigs, mice, raccoons, chickens, ducks, sheep, foxes, mink, and cattle. Figure 9 The image shows the PCR amplification results of bovine-specific primers in DNA templates from pigs, mice, raccoons, chickens, ducks, sheep, foxes, minks, and cattle. Figure 10 The image shows the PCR amplification results of different species-specific primer pairs under the condition of DNA template concentration of 1 ng / μL. Figure 11 The image shows the PCR amplification results of different species-specific primer pairs under the condition of DNA template concentration of 0.1 ng / μL. Figure 12 The amplification results of three multiplex PCR grouping systems after primer concentration optimization: pig-mouse-duck, cow-raccoon dog-chicken, and fox-mink-sheep. Figure 13 Amplification results of a multiplex PCR system using a mixed DNA template. Detailed Implementation

[0021] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0023] Example 1: Design of a primer set for identifying meat sources in beef and mutton products This invention selects mitochondrial NADH dehydrogenase subunit 4 ( ND4 The gene is used as a molecular target. This gene shows significant differences between species and high conservation within species. Furthermore, the multi-copy nature of mitochondrial DNA can significantly improve detection sensitivity, making it particularly suitable for processed meat samples that may be degraded.

[0024] The complete coding sequences of the ND4 gene for the above nine species were obtained from the NCBI GenBank database. Their corresponding mitochondrial genome access numbers and gene regions are as follows: cattle (NC_006853.1: 10529..11906), sheep (NC_001941.1: 10175..11552), pigs (NC_000845.1: 11359..12736), chickens (NC_053523.1:10264..11641), ducks (NC_009684.1: 11302..12679), foxes (NC_008434.1: 10203..11581), raccoon dogs (NC_013700.1: 10210..11587), and mink (NC_020641.1: (10180..11557) and mouse (NC_086031.1:48205162..48212928). Multiple sequence alignment was performed on the above sequences using MEGA software to systematically identify specific SNP sites and internal conserved regions in the ND4 gene of each species. The core criterion was that the selected primer target regions must be completely conserved within the target species, and key base mismatches must exist at corresponding positions in all non-target species (especially closely related species).

[0025] Based on the specific regions identified by comparative analysis, candidate primers were designed using Primer Premier software. The design followed these principles: primer length 18-25 bp, annealing temperature (Tm) 55-65℃, GC content 40%-60%, and avoidance of significant secondary structures. Simultaneously, the amplified fragment lengths for each species were pre-defined to be differentiated between 100-300 bp to facilitate clear distinction in subsequent agarose gel electrophoresis analysis. To theoretically verify the primer specificity, the candidate primers were bioinformatically evaluated using the NCBIBLAST tool. This analysis aimed to confirm the specific binding of the primers to the target ND4 gene sequence and exclude the possibility of cross-reactions with the genomes of non-target species (especially closely related species). The primer sets shown in Table 1 were obtained and synthesized by Jilin Kumei Biotechnology Co., Ltd., and purified using PAGE to ensure amplification efficiency.

[0026] Table 1

[0027] Example 2: Application of a primer set for identifying the meat source of beef and mutton products in detecting the meat source of beef and mutton products. S1: Extract genomic DNA from the sample to be tested. The samples used in this embodiment were sourced from the following sources: one sample each of beef, mutton, pork, chicken, and duck, purchased from Harbin Ha'an Market; one sample each of mink, raccoon dog, and fox meat, purchased from Harbin Hualong Blue Fox Breeding Co., Ltd.; and one sample of rat meat, provided by the Wildlife and Plant Testing Center of the National Forestry and Grassland Administration. All samples were kept fresh during collection and stored at -20℃ after collection. DNA was extracted using a commercial genomic DNA extraction kit (purchased from Suzhou Youyilandi Biotechnology Co., Ltd.), and the sample DNA concentration was determined to be ≥50 ng / μL. S2: Using the DNA extracted in S1 as a template, PCR amplification was performed using the primer set obtained in Example 1 to obtain amplification products. The total volume of the PCR amplification system was 20 μL, and its specific composition was as follows: 10× PCR buffer (Mg-free). 2+ The following solutions were prepared: 2.0 μL of 10 mM dNTP mixture, 0.4 μL of 25 mM MgCl2, 0.20 μL of hot-start Taq polymerase (5 U / μL), 0.4 μL each of forward and reverse primers (10 μM), 1.0 μL of template DNA, and ultrapure water to a final volume of 20 μL. The PCR amplification program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 57℃ annealing for 15 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 5 min. S3: The amplification products obtained in S2 were detected by 3% agarose gel electrophoresis, and the electrophoresis band results were recorded. The meat source was identified based on the electrophoresis band results. If the amplified fragment length is 132 bp, then the meat source of the sample to be tested is cattle; If the amplified fragment length is 250 bp, then the meat source of the sample to be tested is sheep; If the amplified fragment length is 120 bp, then the meat source of the sample to be tested is pig; If the length of the amplified fragment is 193 bp, then the meat source of the sample to be tested is chicken; If the amplified fragment length is 187 bp, then the meat source of the sample to be tested is duck; If the amplified fragment length is 142 bp, then the meat source of the sample to be tested is fox. If the amplified fragment length is 154 bp, then the meat source of the sample to be tested is raccoon dog; If the amplified fragment length is 179 bp, then the meat source of the sample to be tested is mink; If the amplified fragment length is 153 bp, then the meat source of the sample to be tested is mouse.

[0028] The specific steps for agarose gel electrophoresis are as follows: S4: Preparation of 3% agarose gel: Weigh 1.5 g of agarose powder and place it in an Erlenmeyer flask. Add 50 mL of 1×TAE buffer and microwave until the agarose is completely dissolved to obtain a clear and transparent solution. After the solution cools to about 60°C, add GelRed nucleic acid dye to a final concentration of 0.5 μg / mL and mix gently. Pour the mixture into a gel casting plate with a comb in place and let it stand at room temperature for about 20 min until it is completely solidified. Carefully remove the comb vertically and place the gel in the electrophoresis tank. S5: Sample preparation and loading: Use a micropipette to take 5 μL of PCR product from each sample and mix it thoroughly with 1 μL of 6×DNA loading buffer. Add the entire mixture to the gel wells and add 5 μL of 500 bp DNA molecular weight standard (Takara, Japan) to the adjacent wells. S6: Electrophoresis run: Add sufficient 1×TAE electrophoresis buffer to the electrophoresis tank to ensure that the liquid surface completely submerges the gel, set the voltage to 80 V, and electrophoresis for 45 min; S7: Gel Imaging and Analysis: After electrophoresis, the gel is placed in a UV transmission imager to observe the bands and acquire images.

[0029] Electrophoresis results as follows Figures 1-9As shown, the specific primer pairs for cattle, sheep, pigs, chickens, ducks, foxes, raccoons, minks, and mice provided by this invention (nucleotide sequences shown in SEQ ID NO. 1-18) amplified single, clear bands of the expected size in the corresponding target species DNA templates; while no amplified bands, non-specific bands, or primer dimers were observed in any non-target species templates.

[0030] Therefore, the specific primer pairs for cattle, sheep, pigs, chickens, ducks, foxes, raccoons, minks, and mice provided by this invention all have excellent intraspecific amplification efficiency and strict interspecific specificity. Their performance is stable and reliable, and they fully meet the requirements for subsequent construction of multiplex PCR systems and application detection.

[0031] Effect Experiment: 1. PCR sensitivity detection Genomic DNA was extracted from the sample to be tested, and the concentration of the DNA was set to 1 ng / μL and 0.1 ng / μL. Using the DNA extracted in S1 as a template, PCR amplification was performed using the primer set obtained in Example 1 (the PCR amplification system and procedure are the same as above) to obtain amplification products. The amplification products were detected by 3% agarose gel electrophoresis (the steps are the same as above), and the electrophoresis band results were recorded. The meat source was identified based on the electrophoresis band results.

[0032] Electrophoresis results as follows Figure 10 and Figure 11 As shown, when the template DNA concentration was 1 ng / μL, all target species amplified specific bands of the expected size, with clear and uniform brightness. When the template concentration was further diluted to 0.1 ng / μL, clear and bright specific bands could still be detected in cattle, pigs, chickens, ducks, foxes, minks, and mice, while the band signals of sheep and raccoons were weakened, but they were still in the expected positions, and there was no non-specific amplification or interference from other bands.

[0033] Therefore, the species-specific primers provided by this invention still have high detection sensitivity and good amplification stability under low concentration DNA template conditions (as low as 0.1 ng / μL), which can effectively meet the detection needs of trace DNA targets in actual complex samples.

[0034] Species-specific validation of the multiplex PCR system involved dividing the DNA to be tested (0.1 ng / μL) into three groups and mixing them in equal volumes: Group 1: pig-mouse-duck; Group 2: cattle-raccoon dog-chicken; Group 3: fox-mink-sheep. Subsequently, a separate multiplex PCR reaction system was prepared for each group of mixed DNA, and amplification was performed using only the specific primers corresponding to that group's species. The Group 1 reaction system contained only pig, mouse, and duck primers; the Group 2 reaction system contained only cattle, raccoon dog, and chicken primers; and the Group 3 reaction system contained only fox, mink, and sheep primers.

[0035] The PCR amplification systems and procedures described in the three groups are the same as above, except that the volume of upstream and downstream primers (10 μM) added for sheep and raccoon species is changed to 0.8 μL (final concentration 0.4 μM), while the volume of upstream and downstream primers (10 μM) added for pig, rat, duck, cow, chicken, fox, and mink species remains at 0.4 μL (final concentration 0.2 μM). The amplified products obtained above are detected by 3% agarose gel electrophoresis (steps as above), and the electrophoresis band results are recorded. The meat source is identified based on the electrophoresis band results.

[0036] like Figure 12 As shown, the electrophoresis results of the amplification products for the three mixed DNA groups (pig-mouse-duck, cow-raccoon dog-chicken, fox-mink-sheep) showed that in each mixed sample, only the expected band corresponding to the species contained in that group was amplified. For example, in the cow-raccoon dog-chicken mixed DNA sample, only the specific bands of cow, raccoon dog, and chicken were visible, and no cross-amplification or non-specific signals were observed between different species. This indicates that the multiplex PCR detection system provided by this invention can still achieve stable species-specific amplification even under low template concentration (0.1 ng / μL) and mixed DNA from multiple species, and has high detection reliability. In addition, by increasing the final primer concentration for sheep and raccoon dog to 0.4 μM, the band brightness was significantly improved compared to before optimization. Figure 11 The amplification efficiency and system balance were significantly enhanced.

[0037] 3. Validation of the application of multiplex PCR system in complex doped samples To simulate the highly complex multi-species contamination scenarios in actual testing, DNA samples from nine species (cattle, sheep, pigs, chickens, ducks, foxes, raccoon dogs, mink, and mice) were integrated. 5 μL of DNA solution from each species at a concentration of 0.1 ng / μL was mixed to prepare a composite DNA template. Given the susceptibility of DNA degradation in processed meat products, this invention controlled all amplified fragments within a short range of 100-250 bp during primer design. However, this also resulted in similar fragment lengths for some species, making them indistinguishable on agarose gel electrophoresis due to band co-migration if detected simultaneously in a single system. Therefore, this invention employs a grouped detection strategy, scientifically allocating species-specific primers to three independent multiplex PCR systems: ① pig-mouse-duck group, ② cattle-raccoon dog-chicken group, and ③ fox-mink-sheep group. Optimized PCR reaction conditions (with sheep and raccoon dog primers at a final concentration of 0.4 μM, and primers for the other species at a final concentration of 0.2 μM) were used to amplify the composite template. The amplified products obtained above were detected by 3% agarose gel electrophoresis (steps as above), and the electrophoretic band results were recorded. The meat source was identified based on the electrophoretic band results.

[0038] Electrophoresis results ( Figure 13 The results showed that all three amplification systems successfully detected the expected bands from the mixed DNA template, with good band separation within each system and no cross-linking or non-specific amplification. This grouping scheme, while ensuring compatibility with degraded DNA (short fragment design), effectively eliminates fragment interference problems in multi-target co-detection. This allows the present invention to achieve accurate and rapid interpretation of complex samples using only conventional agarose gel electrophoresis, significantly improving the applicability and reliability of the technology in grassroots and practical regulatory scenarios.

[0039] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A primer set for identifying the meat source of beef and mutton products, characterized in that, The primer set includes specific primer pairs for cattle, sheep, pigs, chickens, ducks, foxes, raccoons, minks, and mice; The upstream primer sequence for the bovine sample is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.

2. The upstream primer sequence for the sheep is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.

4. The upstream primer sequence for the pig is shown in SEQ ID NO.5, and the downstream primer sequence is shown in SEQ ID NO.

6. The upstream primer sequence for the chicken is shown in SEQ ID NO.7, and the downstream primer sequence is shown in SEQ ID NO.

8. The upstream primer sequence for the duck is shown in SEQ ID NO.9, and the downstream primer sequence is shown in SEQ ID NO.

10. The upstream primer sequence for the fox is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQ ID NO.

12. The upstream primer sequence for the raccoon dog is shown in SEQ ID NO.13, and the downstream primer sequence is shown in SEQ ID NO.

14. The upstream primer sequence of the mink is shown in SEQ ID NO.15, and the downstream primer sequence is shown in SEQ ID NO.16; The upstream primer sequence for the mouse is shown in SEQ ID NO.17, and the downstream primer sequence is shown in SEQ ID NO.

18.

2. A reagent kit for identifying the meat source of beef and mutton products, characterized in that, The kit contains the primer set as described in claim 1.

3. The application of the primer set according to claim 1 and the kit according to claim 2 in the detection of beef and mutton products.

4. A method for identifying the meat source of beef and mutton products, characterized in that, The method includes the following steps: S1: Extract genomic DNA from the sample to be tested; S2: Using the DNA extracted in S1 as a template, perform multiplex PCR amplification using the primer set described in claim 1 to obtain the amplification product; S3: Perform electrophoretic detection on the amplification products obtained in S2, and identify the meat source based on the detection results.

5. The method according to claim 4, characterized in that, The multiplex PCR amplification described in S2 includes three amplification systems: the first system consists of pigs, mice, and ducks; the second system consists of cattle, raccoons, and chickens; and the third system consists of foxes, minks, and sheep.

6. The method according to claim 4, characterized in that, The multiplex PCR amplification system described in S2 includes the primer set described in claim 1; the thermostable DNA polymerase is a hot-start Taq DNA polymerase, used at a concentration of 0.02-0.1 U / μL; the deoxyribonucleoside triphosphate (dNTPs) are a mixture of dATP, dTTP, dCTP, and dGTP, with each dNTP in the mixture having a final concentration of 100-400 μM; Mg 2+ The ions were provided by magnesium salts at a final concentration of 1.5–3.0 mM; the reaction buffer was Mg-free. 2+ The 10×PCR buffer should be diluted to a working concentration before use.

7. The method according to claim 6, characterized in that, The concentrations of primer sets in the multiplex PCR detection system are as follows: the final concentration of the specific primer pairs for cattle, pigs, chickens, ducks, foxes, minks, and mice is 0.2 μM; the final concentration of the specific primer pairs for sheep and raccoon dogs is 0.4 μM.

8. The method according to claim 4, characterized in that, The conditions for multiplex PCR amplification described in S2 are: 95℃ pre-denaturation for 3-10 min; 95℃ denaturation for 20-45 s, 56-60℃ annealing for 10-60 s, 72℃ extension for 20-45 s, for 25-45 cycles; 72℃ extension for 3-10 min.

9. The method according to claim 4, characterized in that, The electrophoretic detection described in S3 is either agarose gel electrophoresis or capillary electrophoresis, and the meat source is determined based on the length of the amplified fragment.

10. The method according to claim 9, characterized in that, The criteria for determining the meat source are as follows: If the amplified fragment length is 132 bp, then the meat source of the sample to be tested is cattle; If the amplified fragment length is 250 bp, then the meat source of the sample to be tested is sheep; If the amplified fragment length is 120 bp, then the meat source of the sample to be tested is pig; If the amplified fragment length is 193 bp, then the meat source of the sample to be tested is chicken; If the amplified fragment length is 187 bp, then the meat source of the sample to be tested is duck; If the amplified fragment length is 142 bp, then the meat source of the sample to be tested is fox; If the amplified fragment length is 154 bp, then the meat source of the sample to be tested is raccoon dog; If the amplified fragment length is 179 bp, then the meat source of the sample to be tested is mink; If the amplified fragment length is 153 bp, then the meat source of the sample to be tested is mouse.

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