A primer combination, kit, method and application thereof for identifying common edible meat species

Abstract

The present invention discloses a primer combination, a kit, a method, and applications thereof for identifying the species of common edible meats, belonging to the field of molecular identification technology. The primer combination comprises a specific primer pair A for amplifying the bovine 16S rRNA gene, a specific primer pair B for amplifying the sheep COI gene, a specific primer pair C for amplifying the chicken Cytb gene, a specific primer pair D for amplifying the pig COI gene, a specific primer pair E for amplifying the goose NADH2 gene, and a specific primer pair F for amplifying the duck 16S rRNA gene. The nucleotide sequences of these primers are shown in SEQ ID NOs. 1-12. The present invention establishes a forensic detection method and kit with high sensitivity, good specificity, and strong stability, providing an effective detection tool for identifying the species of common edible meats, which is of great significance to forensic identification of meat and its products in my country.

Description

Technical Field

[0001] The present invention relates to the field of molecular identification technology, and in particular to a primer combination, a kit, a method and applications thereof for identifying common edible meat species. Background Art

[0002] Traditional methods for detecting and identifying meat adulteration primarily include morphological, histological, and serological methods. However, these methods place high demands on sample quality, requiring samples and tissues to be highly intact. Furthermore, these methods lack specificity, particularly serological methods, which are limited by protein stability and are difficult to apply to samples damaged by heat, degraded, or corrupted. Histological and morphological methods also require a high level of experience from the investigator, leading to a high degree of subjectivity in their assessments, resulting in significant limitations.

[0003] As the primary genetic material of organisms, DNA possesses excellent structural stability and species-specific genetic information, making it an ideal molecular marker for species identification. Currently, the primary molecular detection technology used for meat species identification is DNA barcoding, which utilizes a short, standardized DNA fragment ubiquitous in the genome as a molecular marker to achieve rapid, accurate, and automated species identification. Compared with traditional meat adulteration detection techniques, these DNA-based molecular detection technologies have become the primary means of species identification for common edible meats due to their high accuracy, sensitivity, specificity, and traceability. Among these, mitochondrial DNA (mtDNA) has emerged as the most promising molecular marker for species identification due to its unique advantages, including high copy number, small molecular weight, excellent thermal stability, and strong resistance to degradation. Mitochondrial cytochrome b (Cytb) gene, cytochrome c oxidase I (COI) gene, 12S ribosomal RNA (12S rRNA) and 16S ribosomal RNA (16S rRNA) genes, and control region (D-loop region) genes are widely used as DNA barcodes in meat testing.

[0004] However, species identification based on DNA barcoding technology is primarily accomplished using universal primer amplification followed by Sanger sequencing. However, when encountering mixtures of DNA from multiple species, sequencing results can produce mixed DNA profiles that are difficult to interpret. Furthermore, degraded samples may not provide sufficient sequence information for comparison. Compared to using universal primers to amplify DNA fragments with interspecies sequence differences, using species-specific primers for DNA fragment length analysis exhibits higher detection efficiency. Based on interspecies sequence differences, species-specific primers are designed to bind only to target species sequences, generating PCR products of varying lengths. Individual species can be identified by detecting species-specific product lengths without the need for sequence analysis. Furthermore, multiplex PCR, which incorporates primers for multiple species detection in a single reaction system, enables simultaneous identification of multiple target species, offering advantages such as high detection efficiency, low cost, and a high success rate. Compared to sequencing-based DNA barcoding technology, species-specific multiplex PCR detection technology is applicable to DNA mixtures; each species in the mixture can be identified using species-specific PCR products in the multiplex PCR analysis.

[0005] In summary, multiplex PCR based on species-specific primers is a simple, efficient, and low-cost technique that can identify more species in a single reaction and can be analyzed by simple agarose gel electrophoresis, saving time and cost while providing more accurate identification results. Therefore, the development of a species-specific multiplex PCR detection system with strong specificity, high sensitivity, and good reproducibility can accurately identify animal-derived ingredients in common edible meats, which is of great significance for the identification of adulteration of common meats and their products. Summary of the Invention

[0006] The purpose of the present invention is to provide a primer combination, a kit and a method and their application for identifying common edible meat species, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a simple and practical detection method for identifying adulteration of common meat and its products.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a primer combination for identifying common edible meat species, the primer combination comprising a specific primer pair A for amplifying the cattle 16s rRNA gene, a specific primer pair B for amplifying the sheep COI gene, a specific primer pair C for amplifying the chicken Cytb gene, a specific primer pair D for amplifying the pig COI gene, a specific primer pair E for amplifying the goose NADH2 gene, and a specific primer pair F for amplifying the duck 16s rRNA gene;

[0009] The nucleotide sequence of the specific primer pair A is shown in SEQ ID NOs.1-2; the nucleotide sequence of the specific primer pair B is shown in SEQ ID NOs.3-4; the nucleotide sequence of the specific primer pair C is shown in SEQ ID NOs.5-6; the nucleotide sequence of the specific primer pair D is shown in SEQ ID NOs.7-8; the nucleotide sequence of the specific primer pair E is shown in SEQ ID NOs.9-10; and the nucleotide sequence of the specific primer pair F is shown in SEQ ID NOs.11-12.

[0010] The present invention also provides a product for identifying common edible meat species, comprising the above primer combination.

[0011] Furthermore, the product comprises a reagent or a kit.

[0012] The present invention also provides a use of the above primer combination in preparing a kit for identifying common edible meat species.

[0013] The present invention also provides a method for identifying common edible meat species, comprising the following steps:

[0014] Using DNA from common edible meat samples as templates, perform PCR amplification using the aforementioned primer combination or product;

[0015] The amplified products were subjected to gel electrophoresis detection, and the species origin of the common edible meat samples was determined based on the electrophoresis bands.

[0016] Furthermore, the common edible meat samples include cattle, sheep, chicken, pig, goose, duck or mixed samples thereof.

[0017] Furthermore, the PCR amplification reaction system includes: 10 μL 2×Taq PCR MasterMix, 4 μL deionized water, 4 μL primer mixture and 2 μL DNA template.

[0018] Furthermore, the reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 1 minute, for a total of 30 cycles; final extension at 72°C for 10 minutes; and insulation at 4°C.

[0019] Furthermore, the gel electrophoresis detection is performed using 1.5% agarose gel.

[0020] The present invention also provides an application of the above primer combination, the above product, or the above method in identifying common edible meat species.

[0021] The present invention discloses the following technical effects:

[0022] 1. Based on comparative genomics, the present invention independently designs a systematic and reliable species-specific DNA sequence screening method. The species-specific DNA sequences obtained by screening can be used as target sequences for species identification after verification.

[0023] 2. The present invention utilizes species-specific multiplex PCR to simultaneously identify samples of six common edible meats (cattle, sheep, chicken, pig, goose, and duck), and can distinguish multiple meats in mixed samples.

[0024] 3. The target fragments amplified by the present invention are all below 500 bp, which can significantly improve the success rate of PCR amplification of processed meat products.

[0025] 4. The experimental process of the present invention is simple and fast, and can be analyzed by simple agarose gel electrophoresis. In addition, species-specific primers can only amplify species with fixed lengths of corresponding amplified fragments, which can be achieved in current domestic DNA laboratories.

[0026] 5. The present invention has established a forensic detection kit with high sensitivity, good specificity and strong stability, which provides an effective detection tool for the species identification of common edible meats, and is of great significance for judicial identification related to meat and its products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 The following are agarose gel electrophoresis patterns of the six common edible meats and their mixed samples and species-specific studies in Example 2; where M: DL 100 DNA marker; 1: standard control DNA sample; 2: cattle; 3: sheep; 4: chicken; 5: pig; 6: goose; 7: duck; 8: donkey; 9: dog; 10: rabbit; 11: horse; 12: mouse; 13: cat; 14: human; 15: blank control;

[0029] Figure 2 The agarose gel electrophoresis patterns for the sensitivity study of identification of the six common edible meat species in Example 3 are shown; a: cattle; b: sheep; c: chicken; d: pig; e: goose; f: duck; in af, M: DL 100 DNA marker; 1: standard control DNA sample; 2: 2 ng / μL; 3: 1 ng / μL; 4: 0.5 ng / μL; 5: 0.25 ng / μL; 6: 0.125 ng / μL; 7: 0.0625 ng / μL; 8: blank control;

[0030] Figure 3 This is the agarose gel electrophoresis pattern of the simulated mixed sample for identification of the six common edible meat species in Example 4; wherein, M: DL 100 DNA marker; 1: standard control DNA sample; 2: 10%; 3: 5%; 4: 1%; 5: 0.5%; 6: blank control. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The present invention provides a primer combination, kit, method, and application thereof for identifying species of common edible meats. The primer combination includes species-specific amplification primers for six common edible meats; the six species-specific amplification primers include a cattle 16S rRNA gene primer, a sheep COI gene primer, a chicken Cytb gene primer, a pig COI gene primer, a goose NADH2 gene primer, and a duck 16S rRNA gene primer.

[0037] In a preferred embodiment of the present invention, the sequences of the species-specific amplification primers for six common edible meats are as follows:

[0038] Bovine 16s rRNA gene primers: SEQ ID NOs. 1-2; sheep COI gene primers: SEQ ID NOs. 3-4; chicken Cytb gene primers: SEQ ID NOs. 5-6; pig COI gene primers: SEQ ID NOs. 7-8; goose NADH2 gene primers: SEQ ID NOs. 9-10; duck 16s rRNA gene primers: SEQ ID NOs. 11-12.

[0039] In addition, the kit includes the above-mentioned primer combination for identification of common edible meat species, 2×Taq PCR Master Mix and deionized water.

[0040] The present invention will be described in detail and specifically below through specific embodiments and drawings for a better understanding of the present invention. However, the following embodiments do not limit the scope of the present invention.

[0041] In the following examples, conventional methods were used unless otherwise specified, and reagents used were conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0042] Example 1

[0043] This embodiment provides a method for identifying common edible meat species, comprising the following steps:

[0044] 1. Screening of species-specific DNA sequences for common edible meats

[0045] The complete mitochondrial genome sequences of the common edible meat species cattle (NC_006853.1), sheep (NC_001941.1, NC_005044.2), chicken (NC_053523.1), pig (NC_000845.1), goose (NC_011196.1), and duck (NC_009684.1) were downloaded from the GenBank database. Sliding-window analysis of the complete mitochondrial genome sequences was performed using DnaSP v5.10.1 software to identify differential nucleotide sequences between species. Sequence similarity analysis of the initially screened nucleotide sequences was performed using online NucleotideBLAST, and those that met the criteria were selected as species-specific DNA sequences. The inclusion criteria for species-specific DNA sequences were: ① the sequence had sufficient base diversity between species, especially between closely related species; ② the sequence had no base length variation within the same species distributed across different geographic regions; ③ the sequence had comprehensive database information, allowing for access to information on the sequence across species; and ④ the sequence was greater than 200 base pairs, facilitating primer design. The mitochondrial genes corresponding to the selected species-specific DNA sequences were annotated based on gene annotation information in GenBank. During this process, the selected candidate DNA sequences were repeatedly verified. Some candidate sequences were excluded due to poor intraspecies conservation, such as the porcine D-loop gene, which has high base diversity within the same species and is prone to false negative results. Ultimately, different mitochondrial genes from six common edible meat species were selected for kit development: the cattle 16s rRNA gene, the sheep COI gene, the chicken Cytb gene, the porcine COI gene, the goose NADH2 gene, and the duck 16s rRNA gene.

[0046] 2. Design and effectiveness of specific primers for common edible meat species

[0047] Based on the selected species-specific DNA sequences, species-specific primers were designed within conserved sequence regions using Primer 5.0 software. Primer design principles were as follows: ① Primer length should be 15-30 bp, with no more than five consecutive identical bases; ② GC content should be between 40-60%; ③ Primer dimers and hairpin structures should be avoided; ④ Primer annealing temperatures should be kept consistent; and ⑤ Amplification product lengths should be as uniform as possible, ranging from 50 to 500 bp. Species specificity of primers was assessed using the NCBI Primer-BLAST function. Mutual interference between multiple primer pairs was also assessed using Multiplex Manager software. Simulated PCR reactions were then performed using the UCSC In-Silico PCR system on DNA from the corresponding species to verify primer-template binding and amplification. Primers used to construct the multiplex amplification typing system should bind exclusively to the target species DNA and produce single, clear, bright, and bright bands without smearing. The high degree of sequence similarity between closely related species mitochondrial DNA places even higher demands on primer specificity. For example, cattle and sheep belong to the Bovidae family, while ducks and geese belong to the Anatidae family. Amplification using mixed primers or mixed DNA can easily lead to nonspecific amplification, unclear amplified bands, and the absence of target amplified bands. Furthermore, the resolution of liposome gel electrophoresis is between 40 and 50 bp. To achieve optimal separation, the six primer pairs were designed to amplify target fragments with a length difference of greater than 40 bp. This process requires repeated design of new primers until the species-specific primers in the mixture amplify only the target species DNA in the mixed DNA.

[0048] After repeated design and verification, the genetic markers and their corresponding amplification primer sequences, amplification fragment lengths and final reaction concentrations were optimized in this embodiment, as shown in Table 1.

[0049] Table 1 Primer sequences, amplified fragment lengths, and final reaction concentrations for genetic markers in common edible meat species identification test kits

[0050]

[0051] 3. Construction and optimization of a species-specific multiplex PCR amplification system for common edible meats

[0052] The constructed composite amplification typing system was continuously adjusted and optimized for PCR reaction conditions including primer ratio concentration, DNA template amount, annealing temperature, and amplification cycle number to obtain balanced and stable PCR product typing results, realizing composite amplification of genetic markers of 6 common edible meat species.

[0053] In this embodiment, the multiplex PCR amplification system containing genetic markers of 6 common edible meat species is preferably selected as shown in Table 2, wherein DNA needs to be extracted from the blood, saliva, muscle, hair, etc. of the 6 common edible meat species.

[0054] Table 2 Multiplex PCR amplification system for genetic markers of common edible meat species

[0055]

[0056] The reaction system can obtain good results by using the following procedure on various reaction thermal cyclers (such as ABI 9700, ABI 9600, ABI 2720, Bio-RadiCycler, etc.). ABI 9700 was used in this embodiment.

[0057] Reaction procedure: 94°C initial denaturation for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min; final extension at 72°C for 10 min; and incubation at 4°C. The volume of the primer mix (10 μM) in Table 2 is 4 μL, meaning that 4 μL of the primer mix was prepared from six primer pairs using the final primer concentrations listed in Table 1.

[0058] 4. Detection of composite amplification products of common edible meat species

[0059] Prepare an agarose gel at a 1.5% concentration. For agarose gel electrophoresis, mix 5 μL of PCR amplification product with 2 μL of loading buffer. Detect the amplified product by electrophoresis using various agarose gel electrophoresis instruments (e.g., E-Gel precast agarose gel electrophoresis system, Major Science SafeBlue blue photophoresis system, etc.).

[0060] Example 2

[0061] This example uses the method provided in Example 1 to detect DNA samples from 13 species, including cattle, sheep, pigs, chickens, geese, ducks, donkeys, dogs, rabbits, horses, mice, cats, and humans, as well as a standard control DNA sample (a mixture of DNA from cattle, sheep, chickens, pigs, geese, and ducks). The specific operations are as follows:

[0062] 1. Collect muscle samples from 13 species including cattle, sheep, pigs, chickens, geese, ducks, donkeys, dogs, rabbits, horses, mice, cats and humans. Blood, saliva, hair and other samples are also available.

[0063] 2. Use DNeasy Blood & Tissue Kit (Qiagen, Germany) to extract DNA. Refer to the kit instructions for specific operations. dsDNAHS Assay Kit (Thermo Fisher, USA) and DNA was quantified using a 2.0 Fluorometer (Thermo Fisher, USA);

[0064] 3. Mix the DNA templates of cattle, sheep, chicken, pig, goose and duck in a ratio of 1:1:1:1:1:1 to prepare standard control DNA samples. The final concentration of each sample in the standard control DNA sample is 1 ng / μL.

[0065] 4. The above DNA samples and standard control DNA samples were subjected to composite amplification of six genetic markers using the detection method constructed in Example 1.

[0066] The results show that ( Figure 1 ), of the 13 species, except for the 6 target species that amplified the expected product bands, no bands were amplified for the bands corresponding to the remaining 7 non-target species. The results showed that the detection method constructed in Example 1 has good species specificity.

[0067] Example 3

[0068] This example uses the method provided in Example 1 to conduct sensitivity research on the constructed multiple amplification system and its kit. The specific operations are as follows:

[0069] 1. Continuously dilute the DNA templates of the six target species (cattle, sheep, chicken, pig, goose and duck) at a gradient of 2 ng / μL, 1 ng / μL, 0.5 ng / μL, 0.25 ng / μL, 0.125 ng / μL and 0.0625 ng / μL.

[0070] 2. Perform multiplex PCR amplification on the above DNA samples and standard control DNA samples to obtain the minimum detection limit of the constructed multiplex PCR detection system. Each concentration is tested in parallel three times.

[0071] The results show that ( Figure 2 ). As the amount of DNA template decreased, the brightness of the amplified products in the agarose gel gradually decreased. Amplified products from all six species were detectable when the DNA template amount was between 0.0625 ng / μL and 2 ng / μL. These results demonstrate that the detection method and kit constructed in Example 1 have high sensitivity.

[0072] Example 4

[0073] This example uses the method provided in Example 1 to detect the DNA mixture of duck meat and beef to simulate adulteration in actual cases. The specific operation is as follows:

[0074] 1. Thinly slice the duck and beef and place them in a 75°C oven to remove moisture and grind into powder. Add the ground duck meat to the beef in proportions of 0.5%, 1%, 5%, or 10% by weight.

[0075] 2. The total mass of each simulated mixed sample is 100 mg. Mix thoroughly and extract DNA.

[0076] 3. Perform multiplex PCR amplification on the above DNA samples and standard control DNA samples to evaluate the practicality of the constructed multiplex PCR amplification system.

[0077] The results show that ( Figure 3 ), even when the proportion of the mixed sample was as low as 0.5%, that is, the amount of non-beef components was as low as 0.5 mg, non-beef components could still be detected in the mixture. The results show that the kit constructed in Example 1 can meet the requirements of meat adulteration in actual situations.

[0078] As can be seen from the above embodiments, the primer combination, kit and method for identifying common edible meat species described in the present invention provide a reliable and convenient detection method for identifying adulteration of common meat and its products, which helps food regulatory agencies and police to combat illegal activities such as animal-derived food fraud.

[0079] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for identifying common edible meat species, characterized in that: The following steps are involved: DNA from common edible meat samples was used as template and PCR amplification was performed using primer combinations; The amplified products are subjected to gel electrophoresis detection, and the species origin of the common edible meat samples is determined based on the electrophoresis bands; The commonly consumed meat samples are cattle, sheep, chicken, pig, goose, duck or mixed samples thereof; The primer combination comprises a specific primer pair A for amplifying the bovine 16s rRNA gene, a specific primer pair B for amplifying the sheep COI gene, a specific primer pair C for amplifying the chicken Cytb gene, a specific primer pair D for amplifying the pig COI gene, a specific primer pair E for amplifying the goose NADH2 gene, and a specific primer pair F for amplifying the duck 16s rRNA gene; The nucleotide sequence of the specific primer pair A is shown in SEQ ID NOs.1-2; the nucleotide sequence of the specific primer pair B is shown in SEQ ID NOs.3-4; the nucleotide sequence of the specific primer pair C is shown in SEQ ID NOs.5-6; the nucleotide sequence of the specific primer pair D is shown in SEQ ID NOs.7-8; the nucleotide sequence of the specific primer pair E is shown in SEQ ID NOs.9-10; and the nucleotide sequence of the specific primer pair F is shown in SEQ ID NOs.11-12.

2. The method according to claim 1, characterized in that The PCR amplification reaction system includes: 10 μL 2×Taq PCR MasterMix, 4 μL deionized water, 4 μL primer mixture and 2 μL DNA template.

3. The method according to claim 1, characterized in that The PCR amplification reaction procedure is as follows: pre-denaturation at 94°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, for a total of 30 cycles; final extension at 72°C for 10 min; and insulation at 4°C.

4. The method according to claim 1, wherein The gel electrophoresis detection is performed using 1.5% agarose gel.

5. A product for identifying common edible meat species, characterized in that: Comprising the primer combination described in claim 1.

6. The product according to claim 5, characterized in that The product comprises a reagent.

7. Use of the primer combination as claimed in claim 1 in preparing a kit for identifying common edible meat species.

8. Use of the primer combination as claimed in claim 1 or the product as claimed in claim 5 in identifying common edible meat species.

Images