Primer combination for identifying 13 animal-derived components and application thereof

Abstract

The invention discloses a primer combination for identifying 13 animal-derived components and application thereof, the primer combination comprises 18 specific primers, the nucleotide sequences of the primers are shown as SEQ ID NO.1-18, the primer combination can be used for identifying 13 animal-derived components of human, chicken, duck, goose, pigeon, mouse, donkey, horse, pig, dog, sheep, goat and cattle, and the primer combination can be used for identifying 13 animal-derived components of human, chicken, duck, goose, pigeon, mouse, donkey, horse, pig, dog, sheep, goat and cattle. The invention also provides a corresponding detection method and a detection kit or a detection reagent. The technology realizes high-throughput, high-specificity and high-sensitivity detection of the 13 animal-derived components, not only simplifies the operation process and improves the detection efficiency, but also has wide applicability.

Description

Technical Field

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

[0002] The identification technology of meat products covers multiple levels from intuitive feeling, protein level to DNA level. The traditional inspection method relying on sensory organs is subjective, especially when facing processed meat products. Due to the addition of pigments, additives and spices, it is difficult to accurately distinguish them by relying on 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 main carrier of biological genetic information, the base sequence of DNA contains rich genetic information. DNA molecular markers, based on DNA polymorphism, show many advantages compared with other genetic markers.

[0003] Mitochondrial DNA (mtDNA) is highly conserved in eukaryotes and has become a commonly used molecular marker for species identification. Among them, 12S rRNA, 16S rRNA, cytochrome b gene (cyt b), cytochrome c oxidase I gene (COI) and NADH dehydrogenase 6 gene (ND6) are commonly used molecular markers; and mtDNA has the characteristics of strong heat resistance, independence from cell morphology, good interspecies polymorphism, and can show higher accuracy, precision and repeatability. In mtDNA, 12S rRNA and 16S rRNA genes are ideal for designing universal primers and specific probes due to their universality in organisms, functional consistency, and the characteristics of containing conservative and variable sequences. 12S rRNA and 16S rRNA genes are suitable for species identification, genetic diversity and kinship studies, and their sequences have been widely used in the identification of animal-derived ingredients. Recently, multiplex PCR methods for the authenticity identification of various meats have been reported, 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 deficiencies of the current traditional PCR detection, the present invention provides a primer combination for identifying 13 kinds of animal-derived components and its application. The present invention targets mitochondrial ND6, 12S rRNA and 16S rRNA genes, designs species-specific primers in mtDNA 12SrRNA or ND6 genes, designs a common forward primer according to the homologous sequence, designs specific directional primers according to the specific regions of each species, and then performs PCR amplification, and performs detection by a 3730x1 fully automatic gene analyzer, takes the 16S rRNA gene as an internal reference, and performs species identification based on the length specificity of the amplified fragments of the ND6 and 12S rRNA genes. After one PCR amplification, the 13 kinds of animal-derived components can be identified by capillary electrophoresis detection.

[0005] To achieve the above object, the embodiments of the present invention adopt the following technical solutions: In a first aspect, a primer combination for identifying 13 animal-derived ingredients, the primer combination contains 18 specific primers, and the nucleotide sequences thereof are shown in SEQ ID NO.1-18.

[0006] The sequences of the 18 specific primers of the present invention are as follows:

[0007] 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; SEQ ID NO.14 is a specific reverse primer for dogs.

[0008] 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.

[0009] SEQ ID NO.17 is a specific forward primer for the amplification of 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 the amplification of the 16S rRNA gene of cattle, goats, chickens, ducks, geese, pigeons, humans, sheep, pigs, horses, donkeys, dogs, and mice.

[0010] Preferably, the primers are 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.

[0011] Preferably, the animals are humans, chickens, ducks, geese, pigeons, mice, donkeys, horses, pigs, dogs, sheep, goats, and cattle.

[0012] In a second aspect, a kit or detection reagent for identifying 13 animal-derived components, the kit or detection reagent contains the above primer combination.

[0013] In a third aspect, a detection method for identifying 13 animal-derived components, using the DNA of the sample to be tested as a template, using the above primer combination, and performing detection by PCR and capillary electrophoresis, and determining the result according to the electrophoresis result map.

[0014] Preferably, using PCR for detection includes configuring an amplification system, and the amplification system is: 6 μL of PCR MasterMix, 2 μL of the primer combination, 11 μL of nuclease-free pure water, and 1 μL of template DNA.

[0015] Preferably, the PCR Master Mix includes 5 mM ammonium sulfate, 10 mM potassium chloride, 50 mM Tris-HC1 at pH 8.3, 5 mM magnesium ions, 0.8 μg / μL BSA, 4% DMSO, 6% ethylene glycol, 1 mM Na 4 P 2 0 7 and 0.25 mM dNTP, and 0.2 U / μL of hot start Taq enzyme.

[0016] Preferably, the primer concentration is 100 μmol / L for each, and the primer combination is as follows;

[0017] 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 for one cycle, a total of 30 cycles; then hold at 60°C for 20 min, and cool down to 4°C after completion.

[0018] Preferably, the CE detection specific peak height in each result map represents a specific animal-derived component.

[0019] Advantages of the present invention: Multiplex fluorescence PCR combined with capillary electrophoresis technology achieves high-throughput, high-specificity, and high-sensitivity detection of 13 animal-derived ingredients, including humans, chickens, ducks, geese, pigeons, mice, donkeys, horses, pigs, dogs, sheep, goats, and cattle. It not only simplifies the operation process and improves the detection efficiency, but also has a wide range of applicability and can be effectively applied to food safety testing, food adulteration identification, and religious regulations compliance testing in ethnic minority areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0021] Figure 1 .The detection results of goose-specific primers for amplifying species, from top to bottom, the species are goose, pigeon, chicken, and donkey; Figure 2 .The detection results of goose-specific primers for amplifying species, from top to bottom, the species are horse, sheep, cattle, and dog; Figure 3 .The detection results of goose-specific primers for amplifying species, from top to bottom, the species are human, goat, mouse, duck, and pig; Figure 4 .Sequence results of the goose specific primer amplification products after cloning and sequencing; Figure 5 .The test results of DNA samples of various species, from top to bottom, are goose, pigeon, chicken, and donkey. The middle frame is the amplification peak of the universal primer of 16S rRNA of each species (as a control site); Figure 6 .The test results of DNA samples of various species, from top to bottom, are horse, sheep, cattle, and dog. The middle box is the amplification peak of the universal primer of 16S rRNA of each species (as a control site); Figure 7 .The test results of DNA samples of various species, from top to bottom, are human, goat, mouse, duck, and pig. The middle box is the amplification peak of the universal primer of 16S rRNA of each species (as a control site); Figure 8 . The results of the amplification test of goose at different gradient input amounts, from top to bottom, the input amounts are 5ng, 0.5ng, 0.05ng, 0.005ng, and 0.001ng respectively; Fig. 9.Results of chicken and duck meat amplification test, from top to bottom, the test results of raw chicken, cooked chicken, raw duck and cooked duck; Fig.10 . Result graph of amplification test of goose meat and donkey meat, from top to bottom are the test results of raw goose meat, cooked goose meat, raw donkey meat and cooked donkey meat; Fig.11 . Result graph of beef and pork amplification test, from top to bottom, the test results of raw beef, cooked beef, raw pork and cooked pork; Fig.12 .The result chart of pigeon meat amplification test, from top to bottom are the test results of raw pigeon meat and cooked pigeon meat respectively. DETAILED DESCRIPTION

[0022] Those skilled in the art can refer to the content of this article to 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 they are all considered to be included in the present invention. The preparation method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the preparation method and application of this article 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 meanings as those of ordinary skill in the art to which the present invention belongs.

[0023] 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.

[0024] 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.

[0025] As used in the present invention, the terms "comprise", "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 to be 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 of only these embodiments.

[0026] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0027] The terms "approximately" and "substantially" in the present invention represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10% deviation from the indicated value, preferably ±5%.

[0028] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments described in the present invention can be implemented in other sequences than those described or illustrated in the present invention.

[0029] The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably a polymeric molecule, comprising ribonucleic acid, deoxyribonucleic acid or analog units thereof. The nucleic acid may be single-stranded or double-stranded. A single-stranded nucleic acid may be a nucleic acid of 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.

[0030] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The primers in the following examples were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0032] Example 1 Primer design and testing According to the mitochondrial gene (mtDNA) accession numbers of various species published by GeneBank, the mtDNA sequences of 13 species were downloaded from NCBI, and the conserved and variable regions among 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 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 and tentatively designated as the specific primer pairs for geese. The results are as follows: Figure 1-3 shown.

[0033] Furthermore, after amplifying the goose DNA sample using the goose specific primer pair (without the fluorescent label), the target band was recovered and purified using a gel recovery kit, and cloned and sequenced. The sequencing results were compared and analyzed with the NCBI database to further determine the specificity of the goose amplification primers. Figure 4 This is the partial sequencing result of goose.

[0034] 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: Table 1:

[0035] 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 labeled with FAM fluorescence at the 5' end.

[0036] Example 2 Composite amplification system test 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 the primer parts determined in Example 1, and all primers are mixed according to the proportions explored in the experiment 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.

[0037] Table 2:

[0038] This example performs amplification detection on 13 known standard species DNA samples. The specific steps are as follows: 1) Sample preparation DNA samples of standard species were taken, and the concentration and purity of DNA were determined using NanoDrop2000 (Thermo), and the DNA was diluted to the corresponding concentration and stored at 4°C or -20°C for future use; 2) Preparation of amplification system Prepare the PCR amplification system according to the components in Table 3, shake and mix, and then divide into packages according to the number of samples.

[0039] Table 3. PCR amplification system

[0040] 3) Add template Add 1µL of each prepared DNA sample to the corresponding PCR reaction tube. At the same time, set up a negative control: 1µL of nucleic acid-free pure water.

[0041] 4) PCR amplification Place each reaction tube in the reaction tank of the PCR amplification instrument and set the reaction system to 20 µL. Perform PCR amplification according to the following procedure: denaturation at 95°C for 5 min; denaturation at 94°C for 10 s, annealing at 59°C for 90 s for one cycle, for a total of 30 cycles; then maintain at 60°C for 20 min, and cool to 4°C after completion.

[0042] 5) Capillary electrophoresis detection of amplified products Prepare a sample mixture containing molecular weight internal standard and formamide: (0.5μL molecular weight internal standard + 8.5μL formamide) × number of samples to be tested, vortex and mix for 10-15 seconds; use a pipette to dispense 9μL of formamide and internal standard mixture into each test well; take 1μL of amplification product and add it to the formamide and internal standard mixture, and cover with a rubber cap. Perform the test according to the steps in the genetic analyzer user manual.

[0043] 6) Data analysis Import relevant files into GeneMapper software, input the raw data (.fsa file) from the detector, and analyze the data.

[0044] 7) Result determination, like Figure 5-7 As shown, Figure 5-7 The results of capillary detection of 13 species are shown in the figure. The results show that this detection system can accurately identify 13 animal-derived species.

[0045] Example 3 Sensitivity Experiment 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 the gradient detection of geese are shown in Table 4. Table 4 Statistics of detection results of gradient samples of various species:

[0046] 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.

[0047] Example 4 Mixed sample detection 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.

[0048] Table 5:

[0049] The detection was performed according to the steps in Example 2 to determine the detection status of the mixed sample, and the peak height ratio of the spiked species / main species of all combinations was comprehensively counted, as shown in Table 6. The results showed that when the total nucleic acid amount of the cell line DNA was 10 ng, the contamination of other species within the detection range of 1% could be clearly identified.

[0050] Table 6 Statistics of the ratio of species-specific peak height to main species peak height for each species added pairwise:

[0051] Example 5: Heat treatment sample detection In this example, in order to evaluate the effectiveness of heat treatment on meat sample detection, raw meat of seven species, including chicken, duck, goose, donkey, cattle, pigeon, was purchased from the market and treated with braising. Then, DNA was extracted from the raw meat and the cooked meat treated with braising, and the test was carried out according to the steps of Example 2. The results are as follows: Figure 9-12 As 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.

[0052] Advantages of the present invention: Multiplex fluorescence PCR combined with capillary electrophoresis technology achieves high-throughput, high-specificity, and high-sensitivity detection of 13 animal-derived ingredients, including humans, chickens, ducks, geese, pigeons, mice, donkeys, horses, pigs, dogs, sheep, goats, and cattle. It not only simplifies the operation process and improves the detection efficiency, but also has a wide range of applicability and can be effectively applied to food safety testing, food adulteration identification, and religious regulations compliance testing in ethnic minority areas.

[0053] Finally, it is explained that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than 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 recorded 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 implementation method of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with the technology in the 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 shall 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, and the nucleotide sequences thereof are shown as SEQ ID NO.1-18.

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 are 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 described in claim 1 is used, PCR and capillary electrophoresis are used for detection, and the result is determined according to the electrophoresis result map.

6. The detection method according to claim 5, characterized in that: The detection using PCR includes configuring an amplification system, wherein the amplification system is: 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 primers shown in SEQ ID NO.1 is 4.5-5.5 μL; the amount of primers shown in SEQ ID NO.2 is 1.25-1.55 μL; the amount of primers shown in SEQ ID NO.3 is 1.8-2.2 μL; the amount of primers shown in SEQ ID NO.4 is 2.7-3.3 μL; the amount of primers shown in SEQ ID NO.5 is 3.6-4.5 μL; the amount of primers shown in SEQ ID NO.6 is 0.26-0.32 μL; the amount of primers shown in SEQ ID NO.7 is 4.5-5.5 μL; the amount of primers shown in SEQ ID NO.8 is 5.4-6.6 μL; the amount of primers shown in SEQ ID NO.9 is 0.26-0.32 μL; the amount of primers shown in SEQ ID NO.10 is 1.4-1.8 μL; The amount of primer shown in NO.11 added is 0.26-0.32μL; the amount of primer shown in SEQ ID NO.12 added is 3.6-4.5μL; the amount of primer shown in SEQ ID NO.13 added is 0.7-0.9μL; the amount of primer shown in SEQ ID NO.14 added is 0.25-0.3μL; the amount of primer shown in SEQ ID NO.15 added is 2.7-3.3μL; the amount of primer shown in SEQ ID NO.16 added is 2.7-3.3μL; the amount of primer shown in SEQ ID NO.17 added is 0.8-1.2μL; the amount of primer shown in SEQ ID NO.18 added is 0.8-1.2μL.

9. The detection method according to claim 5, characterized in that: The PCR reaction procedure 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.

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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