A primer combination for identifying 13 animal-derived ingredients and its application

Abstract

The present invention discloses a primer combination for identifying 13 animal-derived ingredients and its application. The primer combination comprises 18 specific primers, whose nucleotide sequences are shown in SEQ ID NOs. 1-18. The primer combination can be used to identify 13 animal-derived ingredients from humans, chickens, ducks, geese, pigeons, mice, donkeys, horses, pigs, dogs, sheep, goats, and cattle. Corresponding detection methods and detection kits or reagents are also provided. This technology enables high-throughput, high-specificity, and high-sensitivity detection of these 13 animal-derived ingredients, simplifying the operational process, improving detection efficiency, and having wide applicability.

Description

Technical Field

[0001] The present invention relates to the technical field of animal molecular biology detection, in particular to a primer combination for identifying 13 animal-derived components and an application thereof. Background Art

[0002] Meat product identification technology covers multiple levels, from intuitive perception to protein level to DNA level. Traditional sensory inspection methods are subjective, especially when dealing with processed meat products. Due to the addition of pigments, additives, and spices, it is difficult to accurately distinguish them through sensory inspections such as vision, touch, and smell. Protein-based detection methods are also limited in their widespread application due to factors such as insufficient sensitivity, poor reliability, and high equipment costs. As the primary carrier of biological genetic information, DNA's base sequence contains a wealth of genetic information. DNA molecular markers, based on DNA polymorphism, exhibit numerous advantages over other genetic markers.

[0003] Mitochondrial DNA (mtDNA) is highly conserved among eukaryotes and has become a commonly used molecular marker for species identification. Among these markers, 12S rRNA, 16S rRNA, cytochrome b gene (cyt b), cytochrome c oxidase I gene (COI), and NADH dehydrogenase 6 gene (ND6) are commonly used. mtDNA is also characterized by its high thermostability, independence from cell morphology, and high interspecies polymorphism, resulting in higher accuracy, precision, and reproducibility. Within mtDNA, the 12S rRNA and 16S rRNA genes are ideal for designing universal primers and specific probes due to their ubiquity, functional consistency, and the inclusion of both conserved and variable sequences. The 12S rRNA and 16S rRNA genes are suitable for species identification, genetic diversity, and phylogenetic relationships studies, and their sequences have been widely used to authenticate animal-derived ingredients. Recently, multiplex PCR methods have been reported for meat authenticity testing, covering species such as pigs, cattle, goats, and sheep. However, in actual operation, traditional PCR testing requires multiple steps such as amplification and gel electrophoresis, which is time-consuming, prone to contamination, and easily leads to false positive and false negative results, affecting the sensitivity and accuracy of the test. Summary of the Invention

[0004] In view of the above-mentioned shortcomings of current traditional PCR detection, the present invention provides a primer combination and application for identifying 13 animal-derived components. The present invention targets mitochondrial ND6, 12S rRNA and 16S rRNA genes, designs species-specific primers in the mtDNA 12SrRNA or ND6 gene, designs a common forward primer based on the homologous sequence, and designs specific directional primers based on the specific regions of each species. PCR amplification is then performed and detection is performed using a 3730x1 fully automatic genetic analyzer. Using the 16S rRNA gene as an internal reference, species identification is performed based on the length specificity of the amplified fragments of the ND6 and 12S rRNA genes. After a single PCR amplification, capillary electrophoresis detection can be performed to identify 13 animal-derived components.

[0005] To achieve the above objectives, the embodiments of the present invention adopt the following technical solutions:

[0006] In a first aspect, a primer combination for identifying 13 animal-derived ingredients comprises 18 specific primers, the nucleotide sequences of which are shown in SEQ ID NOs. 1-18.

[0007] The sequences of the 18 specific primers of the present invention are as follows:

[0008]

[0009] Among them, SEQ ID NO.1-13 are primers for detecting the 12S rRNA gene of animals, specifically as follows: SEQ ID NO.1 is a common forward primer for cattle, goats, chickens, ducks, geese, and pigeons; SEQ ID NO.2 is a specific reverse primer for cattle; SEQ ID NO.3 is a specific reverse primer for goats; SEQ ID NO.4 is a specific reverse primer for chickens; SEQ ID NO.5 is a specific reverse primer for ducks; SEQ ID NO.6 is a specific reverse primer for geese; SEQ ID NO.7 is a specific reverse primer for pigeons. SEQ ID NO.8 is a common forward primer for humans, sheep, pigs, horses, donkeys, and dogs; SEQ ID NO.9 is a specific reverse primer for humans; SEQ ID NO.10 is a specific reverse primer for sheep; SEQ ID NO.11 is a specific reverse primer for pigs; SEQ ID NO.12 is a specific reverse primer for horses; SEQ ID NO.13 is a specific reverse primer for donkeys; and SEQ ID NO.14 is a specific reverse primer for dogs.

[0010] SEQ ID NO.15 is a specific forward primer for amplifying the mouse ND6 gene; SEQ ID NO.16 is a specific reverse primer for amplifying the mouse ND6 gene.

[0011] SEQ ID NO.17 is a specific forward primer for amplifying the 16S rRNA gene of cattle, goats, chickens, ducks, geese, pigeons, humans, sheep, pigs, horses, donkeys, dogs, and mice; SEQ ID NO.18 is a specific reverse primer for amplifying the 16S rRNA gene of cattle, goats, chickens, ducks, geese, pigeons, humans, sheep, pigs, horses, donkeys, dogs, and mice.

[0012] Preferably, the primer is fluorescently labeled; preferably, the fluorescent label is located at the 5' end of the common primer, or the 5' end of the forward primer of the universal primer; more preferably, the fluorescent label is a FAM fluorescent label.

[0013] Preferably, the animal is a human, chicken, duck, goose, pigeon, mouse, donkey, horse, pig, dog, sheep, goat or cow.

[0014] In a second aspect, a kit or detection reagent for identifying 13 animal-derived components, the kit or detection reagent contains the above-mentioned primer combination.

[0015] Thirdly, a detection method for identifying 13 animal-derived ingredients uses the DNA of the test object as a template, utilizes the above-mentioned primer combination, uses PCR and capillary electrophoresis detection for detection, and determines the result based on the electrophoresis result map.

[0016] Preferably, the detection using PCR includes configuring an amplification system, wherein the amplification system is: 6 μL of PCR MasterMix, 2 μL of primer combination, 11 μL of nuclease-free pure water, and 1 μL of template DNA.

[0017] Preferably, the PCR Master Mix includes 5 mM ammonium sulfate, 10 mM potassium chloride, 50 mM Tris-HCl (pH 8.3), 5 mM magnesium ion, 0.8 ug / uL BSA, 4% DMSO, 6% ethylene glycol, 1 mM Na4P207 and 0.25 mM dNTP, and 0.2 U / uL hot start Taq enzyme.

[0018] Preferably, the primer concentration is 100 μmol / L, and the primer combination is as follows;

[0019]

[0020] Preferably, the PCR reaction program is: denaturation at 95°C for 5 min; denaturation at 94°C for 10 s, annealing at 59°C for 90 s as one cycle, for a total of 30 cycles; then maintaining at 60°C for 20 min, and cooling to 4°C after completion.

[0021] Preferably, the CE detection-specific peak height in each result spectrum represents a specific animal-derived component.

[0022] Advantages of this invention: Multiplex fluorescence PCR combined with capillary electrophoresis enables high-throughput, high-specificity, and high-sensitivity detection of 13 animal-derived ingredients: human, chicken, duck, goose, pigeon, mouse, donkey, horse, pig, dog, sheep, goat, and cattle. This method not only simplifies the operational process and improves detection efficiency, but also has broad applicability, including effective applications in food safety testing, identification of food adulteration, and compliance with religious regulations in ethnic minority areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, 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.

[0024] Figure 1 .The detection results of the species amplified by the goose-specific primer pair, from top to bottom, are goose, pigeon, chicken, and donkey;

[0025] Figure 2 .The detection results of the species amplified by the goose-specific primer pair, from top to bottom, are horse, sheep, cattle, and dog;

[0026] Figure 3 .The detection results of the species amplified by the goose-specific primer pair, from top to bottom, are human, goat, mouse, duck, and pig;

[0027] Figure 4 Sequence results of the goose-specific primer amplification products after cloning and sequencing;

[0028] Figure 5 DNA sample test results for each species (from top to bottom): goose, pigeon, chicken, and donkey. The center box indicates the amplification peak of the universal 16S rRNA primer for each species (serving as a control site).

[0029] Figure 6 DNA sample test results for each species (from top to bottom): horse, sheep, cattle, and dog. The center box indicates the amplification peak of the universal 16S rRNA primer for each species (serving as a control site).

[0030] Figure 7 DNA sample test results for each species (from top to bottom): human, goat, mouse, duck, and pig. The center box indicates the amplification peak of the universal 16S rRNA primer for each species (serving as a control site).

[0031] Figure 8. Graph of amplification test results of goose at various gradient input amounts, from top to bottom: 5ng, 0.5ng, 0.05ng, 0.005ng, 0.001ng;

[0032] Figure 9 Amplification test results for chicken and duck meat, showing, from top to bottom, the results for raw chicken, cooked chicken, raw duck, and cooked duck.

[0033] Figure 10 Amplification test results for goose and donkey meat, showing, from top to bottom, the results for raw goose, cooked goose, raw donkey, and cooked donkey meat.

[0034] Figure 11 Amplification test results for beef and pork, showing, from top to bottom, the results for raw beef, cooked beef, raw pork, and cooked pork.

[0035] Figure 12 .The result spectrum of pigeon meat amplification test, from top to bottom are the test results of raw pigeon meat and cooked pigeon meat respectively. DETAILED DESCRIPTION

[0036] Those skilled in the art can refer to the contents of this article and realize its application. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The preparation method and application of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the preparation method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0037] The following terms or definitions are provided merely to aid understanding of the present invention. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0038] Unless otherwise defined hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the present invention are intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0039] As used herein, the terms "comprises," "comprising," "having," "containing," or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising." If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of these embodiments.

[0040] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" include a plural of that noun.

[0041] The terms "approximately" and "substantially" in the present invention represent an accuracy range that can be understood by those skilled in the art and can still ensure the technical effect of the characteristics discussed. This term usually means ±10%, preferably ±5%, of the deviation from the indicated value.

[0042] Furthermore, the terms first, second, third, (a), (b), (c), and the like, in the description and claims, are used to distinguish between similar elements and are not necessarily intended to describe a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments described herein can be practiced in other sequences than described or illustrated herein.

[0043] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, comprising ribonucleic acid, deoxyribonucleic acid, or analogs thereof. The nucleic acid may be single-stranded or double-stranded. A single-stranded nucleic acid may be a nucleic acid that is one strand of a denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid may be a single-stranded nucleic acid that is not derived from any double-stranded DNA.

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0045] The primers in the following examples were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0046] Example 1 Primer design and testing

[0047] According to the accession numbers of mitochondrial genes (mtDNA) of various species published by GeneBank, the mtDNA sequences of 13 species were downloaded from NCBI, and the conserved and variable regions between species were compared and analyzed using ApE software. Combined with BLAST analysis, species-specific primers and universal primers were designed. Specific primers for cattle, goats, chickens, ducks, geese, pigeons, humans, sheep, pigs, horses, donkeys, and dogs were designed on the 12S rRNA and ND6 genes. Universal primers were used for internal control and were designed on the 12S rRNA. The specificity of the primer pairs was further tested by single PCR and cross-amplification. Among them, taking the screening of specific primers for geese as an example, the specific primer pairs of goose species were used, and the standard DNA samples of each species were used as templates for amplification detection. Finally, the primer pairs that were amplified only in goose samples and had no obvious amplification in samples of other species were selected. They were tentatively designated as specific primer pairs for geese. The results are as follows: Figure 1-3 shown.

[0048] Furthermore, goose DNA samples were amplified using the aforementioned goose-specific primer pair (minus the fluorescent label). The target bands were recovered and purified using a gel extraction kit and cloned for sequencing. The sequencing results were compared with the NCBI database to further confirm the specificity of the goose amplification primers. Figure 4 Partial sequencing results for goose.

[0049] Based on the above method, the primers for each site were tested and screened, and the sequence of the primer combination of the present invention was finally determined, as shown in Table 1:

[0050] Table 1:

[0051]

[0052] The common primers were all labeled with FAM fluorescence at the 5' end, and the forward primers of the mouse-specific primers and the control site primers common to 13 species were also labeled with FAM fluorescence at the 5' end.

[0053] Example 2: Test of the composite amplification system

[0054] The composite amplification detection system of this embodiment includes PCR Master Mix, internal standard, etc. The main components of PCR Master Mix include hot start Taq enzyme, amplification buffer, etc. The primers are as described in Example 1, and all primers are mixed according to the experimental ratio to prepare Primer Mix. The PCR reaction system adopts a 20μL basic system, in which the primer mix is ​​a primer concentration of 100μmol / L, and the primer combination is shown in Table 2.

[0055] Table 2:

[0056]

[0057] This example performs amplification testing on 13 known standard species DNA samples. The specific steps are as follows:

[0058] 1) Sample preparation

[0059] DNA samples of standard species were collected, and the concentration and purity of the DNA were determined using NanoDrop2000 (Thermo). The DNA was then diluted to the corresponding concentration and stored at 4°C or -20°C until use.

[0060] 2) Preparation of amplification system

[0061] Prepare the PCR amplification system according to the components in Table 3, shake and mix thoroughly, and then divide the system into smaller pieces according to the number of samples.

[0062] Table 3. PCR amplification system

[0063]

[0064] 3) Add template

[0065] Add 1µL of each prepared DNA sample to the corresponding PCR reaction tube. At the same time, set up a negative control: 1µL of nuclease-free pure water.

[0066] 4) PCR amplification

[0067] Place each reaction tube into the reaction chamber of a PCR amplifier and set the reaction volume to 20 µL. Perform PCR amplification according to the following protocol: denaturation at 95°C for 5 minutes, followed by denaturation at 94°C for 10 seconds and annealing at 59°C for 90 seconds, for a total of 30 cycles; then, hold at 60°C for 20 minutes, then cool to 4°C.

[0068] 5) Capillary electrophoresis detection of amplified products

[0069] Prepare a sample loading mixture containing a molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) x number of samples to be tested. Vortex and mix thoroughly for 10-15 seconds. Use a pipette to dispense 9 μL of the formamide and internal standard mixture into each well. Add 1 μL of the amplified product to the formamide and internal standard mixture and seal the plate with adhesive. Perform the test according to the instructions in the genetic analyzer user manual.

[0070] 6) Data Analysis

[0071] Import relevant files into GeneMapper software, input the raw data (.fsa file) from the detector, and analyze the data.

[0072] 7) Result determination,

[0073] like Figure 5-7 As shown, Figure 5-7 The results of capillary tube testing of 13 species are shown in the figure. The results show that this detection system can accurately identify 13 animal-derived species.

[0074] Example 3 Sensitivity experiment

[0075] In this example, five gradients (5 ng, 0.5 ng, 0.05 ng, 0.005 ng, 0.001 ng) of templates were prepared for DNA samples of various species, and the detection was performed according to the steps in Example 2 to determine the concentration range of the detection, where Figure 8 The results of each gradient test for geese are shown in Table 4.

[0076] Table 4 Statistics of detection results of gradient samples of various species:

[0077]

[0078] The test results showed that when the template input amount was 0.005ng, all species could be effectively detected; when the input amount was 0.001ng, samples of all species except duck and horse could be effectively detected.

[0079] Example 4 Mixed sample detection

[0080] In this example, the standard DNAs of 13 species were diluted to 10 ng / μL, and then the DNAs were mixed in pairs according to Table 5.

[0081] Table 5:

[0082]

[0083] Testing was performed according to the steps in Example 2 to determine the detection status of the mixed sample. The ratio of the spiked species peak height to the main species peak height for all combinations was comprehensively calculated and shown in Table 6. The results demonstrated that when the total nucleic acid content of the cell line DNA was 10 ng, contamination from other species within the detection range of 1% could be clearly identified.

[0084] Table 6 Statistics of the ratio of species-specific peak height to main species peak height of each species spiked pairwise:

[0085]

[0086] Example 5: Heat Treatment Sample Detection

[0087] In this example, to evaluate the effectiveness of heat treatment on meat sample detection, raw meat of seven species, including chicken, duck, goose, donkey, cattle, pigeon, and pigeon, was purchased from the market and then subjected to both untreated and braising treatments. DNA was then extracted from the untreated raw meat and the braising-treated cooked meat, and the test was performed according to the steps of Example 2. The results are shown in Figure 2. Figure 9-12As shown, specific product peaks of chicken, duck, goose, donkey, cattle, pig and pigeon can be amplified, indicating that the present invention is suitable for identifying animal-derived components in processed meat products.

[0088] Advantages of this invention: Multiplex fluorescence PCR combined with capillary electrophoresis enables high-throughput, high-specificity, and high-sensitivity detection of 13 animal-derived ingredients: human, chicken, duck, goose, pigeon, mouse, donkey, horse, pig, dog, sheep, goat, and cattle. This method not only simplifies the operational process and improves detection efficiency, but also has broad applicability, including effective applications in food safety testing, identification of food adulteration, and compliance with religious regulations in ethnic minority areas.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the above embodiments, it should be understood in the art that the technical solutions described in the above embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited to this. Any changes or replacements that can be easily thought of by any technician familiar with the technology in this field within the technical scope disclosed in the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A primer combination for identifying 13 animal-derived ingredients, characterized in that: The primer combination contains 18 specific primers, whose nucleotide sequences are shown in SEQ ID NO.1-18, wherein SEQ ID NO.1 is a common forward primer for cattle, goats, chickens, ducks, geese, and pigeons; SEQ ID NO.2 is a specific reverse primer for cattle; SEQ ID NO.3 is a specific reverse primer for goats; SEQ ID NO.4 is a specific reverse primer for chickens; SEQ ID NO.5 is a specific reverse primer for ducks; SEQ ID NO.6 is a specific reverse primer for geese; and SEQ ID NO.7 is a specific reverse primer for pigeons. SEQ ID NO.8 is a common forward primer for humans, sheep, pigs, horses, donkeys, and dogs; SEQ ID NO.9 is a specific reverse primer for humans; SEQ ID NO.10 is a specific reverse primer for sheep; SEQ ID NO.11 is a specific reverse primer for pigs; SEQ ID NO.12 is a specific reverse primer for horses; SEQ ID NO.13 is a specific reverse primer for donkeys; SEQ ID NO.14 is a specific reverse primer for dogs; SEQ ID NO.15 is a specific forward primer for amplifying the ND6 gene of mice; SEQ ID NO.16 is a specific reverse primer for amplifying the ND6 gene of mice; SEQ ID NO.17 is a specific forward primer for amplifying the 16S rRNA gene of cattle, goats, chickens, ducks, geese, pigeons, humans, sheep, pigs, horses, donkeys, dogs, and mice; SEQ ID NO.18 is a specific reverse primer for amplifying the 16S rRNA gene of cattle, goats, chickens, ducks, geese, pigeons, humans, sheep, pigs, horses, donkeys, dogs and mice.

2. The primer combination according to claim 1, characterized in that The primer is fluorescently labeled; the fluorescent label is located at the 5' end of the common primer or the 5' end of the forward primer of the universal primer; the fluorescent label is a FAM fluorescent label.

3. The primer combination according to claim 1, characterized in that The animals include humans, chickens, ducks, geese, pigeons, mice, donkeys, horses, pigs, dogs, sheep, goats and cows.

4. A kit or detection reagent for identifying 13 animal-derived ingredients, characterized in that: The kit or detection reagent contains the primer combination according to claim 1.

5. A method for identifying 13 animal-derived ingredients, characterized in that: The DNA of the test object is used as a template, the primer combination according to claim 1 is used, PCR and capillary electrophoresis are used for detection, and the result is determined according to the electrophoresis result pattern.

6. The detection method according to claim 5, characterized in that Detection using PCR includes configuring an amplification system, which includes: 6 μL of PCR Master Mix, 2 μL of primer combination, 11 μL of nuclease-free pure water, and 1 μL of template DNA.

7. The detection method according to claim 6, characterized in that PCR Master Mix includes 5mM ammonium sulfate, 10mM potassium chloride, 50mM Tris-HCl (pH 8.3), 5mM magnesium ion, 0.8ug / uL BSA, 4% DMSO, 6% ethylene glycol, 1mM Na4P207 and 0.25mM dNTP, and 0.2U / uL hot start Taq enzyme.

8. The detection method according to claim 6, characterized in that The primer concentration is 100 μmol / L. The amount of primer shown in SEQ ID NO.1 is 4.5-5.5 μL; the amount of primer shown in SEQ ID NO.2 is 1.25-1.55 μL; the amount of primer shown in SEQ ID NO.3 is 1.8-2.2 μL; the amount of primer shown in SEQ ID NO.4 is 2.7-3.3 μL; the amount of primer shown in SEQ ID NO.5 is 3.6-4.5 μL; the amount of primer shown in SEQ ID NO.6 is 0.26-0.32 μL; the amount of primer shown in SEQ ID NO.7 is 4.5-5.5 μL; the amount of primer shown in SEQ ID NO.8 is 5.4-6.6 μL; the amount of primer shown in SEQ ID NO.9 is 0.26-0.32 μL; The amount of primer shown in SEQ ID NO.10 added is 1.4-1.8 μL; the amount of primer shown in SEQ ID NO.11 added is 0.26-0.32 μL; the amount of primer shown in SEQ ID NO.12 added is 3.6-4.5 μL; the amount of primer shown in SEQ ID NO.13 added is 0.7-0.9 μL; the amount of primer shown in SEQ ID NO.14 added is 0.25-0.3 μL; the amount of primer shown in SEQ ID NO.15 added is 2.7-3.3 μL; the amount of primer shown in SEQ ID NO.16 added is 2.7-3.3 μL; the amount of primer shown in SEQ ID NO.17 added is 0.8-1.2 μL; and the amount of primer shown in SEQ ID NO.18 added is 0.8-1.2 μL.

9. The detection method according to claim 5, characterized in that The PCR reaction procedure was as follows: denaturation at 95° C. for 5 min; denaturation at 94° C. for 10 s, and annealing at 59° C. for 90 s, for a total of 30 cycles; then maintaining at 60° C. for 20 min, and cooling to 4° C. after completion.

10. The detection method according to claim 5, characterized in that: The CE-detection-specific peak height in each result profile represents a specific animal-derived component.

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