Primer group, kit and detection method for simultaneously identifying 12 animal-derived components based on mitochondrial genes

Abstract

The invention discloses a primer group, a kit and a detection method for simultaneously identifying 12 animal-derived components based on mitochondrial genes, and belongs to the technical field of species identification. The primer group comprises primer pairs for respectively amplifying a goat D-LOOP gene, a cattle 16S rRNA gene, a donkey NADH2 gene, a chicken CYTB gene, a dog ATP8 gene, a pig COXI gene, a horse ATP6 gene, a sheep NADH4 gene, a goose 16S rRNA gene, a mouse NADH4 gene, a duck NADH6 gene and a cat NADH2 gene. The primer group, the kit and the detection method capable of simultaneously amplifying and identifying the 12 animal-derived components are constructed based on the mitochondrial genes, a reliable and convenient detection means is provided for forensic medicine and animal food identification, and the primer group, the kit and the detection method have important significance on rapid identification of common animal species.

Description

Technical Field

[0001] The present invention relates to the technical field of species identification, and in particular to a primer set, a kit and a detection method for simultaneously identifying 12 kinds of animal-derived components based on mitochondrial genes. Background Art

[0002] Species identification refers to the identification of the species source of biological samples (such as body fluids, secretions, hair, muscles, bones, etc.), that is, to determine whether the sample belongs to the human body or a certain animal. In addition to being used to analyze and identify biological samples left at the crime scene, and to provide important clues for case investigation and on-site reconstruction, species identification also plays an important role in the field of food safety. Due to the economic benefits brought by the price difference of different meats, criminals will sell cheap meats such as pork, chicken, and duck, or even inedible meats (rat meat, etc.) instead of high-priced meats such as beef, mutton, and donkey meat. Meat adulteration not only disrupts market order and causes economic losses, but may also affect the health of consumers and even trigger ethnic and religious conflicts. Therefore, species identification of unknown biological samples is an important task for forensic workers and a basic problem that needs to be solved in the field of forensic science.

[0003] As the main genetic material of organisms, DNA has good structural stability and species-specific genetic information. Among them, mitochondrial DNA (mtDNA) has become an ideal molecular marker for species identification because of its high copy number, high mutation rate, good stability and low recombination rate compared to nuclear DNA. At present, the use of universal primers for mtDNA gene amplification combined with Sanger sequencing is a conventional method for species identification, but when encountering a mixture of DNA from multiple species, it will produce a mixed DNA map that is difficult to interpret. In addition, degraded samples often cannot provide enough sequence information for comparison. In recent years, species-specific PCR based on mtDNA gene amplification has received widespread attention. This method involves using more than one pair of species-specific primers to amplify different fragments of the mtDNA gene, followed by agarose gel electrophoresis. Compared with conventional PCR, multiplex PCR with species-specific primers has the advantages of simplicity, specificity and simultaneous detection of multiple species. However, the resolution of agarose gel electrophoresis is low, which will limit the number of specific primer pairs in the composite amplification system, resulting in a limited number of species that can be detected. In addition, due to the high similarity of mitochondrial sequences of closely related species, especially species within the same genus, the screening of specific sequences and the design of specific primers are difficult, and the increase in the number of primers in the multiplex PCR system will lead to more cross-reactions, so that the currently constructed species identification detection methods can identify 4-8 target species. Therefore, there is still a lack of accurate, low-cost, and high-throughput common animal species identification detection methods. Summary of the invention

[0004] The purpose of the present invention is to provide a primer set, a kit and a detection method for simultaneously identifying 12 kinds of animal-derived ingredients based on mitochondrial genes, so as to solve the problems existing in the above-mentioned prior art. The present invention constructs a primer set, a kit and a detection method capable of simultaneously amplifying and identifying 12 kinds of animal-derived ingredients based on mitochondrial genes, provides a reliable and convenient detection method for forensic medicine and animal food identification, and is of great significance for the rapid identification of common animals such as goats, cattle, donkeys, chickens, dogs, pigs, horses, sheep, geese, mice, ducks and cats.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a primer set for simultaneously identifying 12 kinds of animal-derived components based on mitochondrial genes, the primer set comprising:

[0007] A primer pair for amplifying goat D-LOOP gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.1-2;

[0008] A primer pair for amplifying the bovine 16S rRNA gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.3-4;

[0009] A primer pair for amplifying the donkey NADH2 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.5-6;

[0010] A primer pair for amplifying chicken CYTB gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.7-8;

[0011] A primer pair for amplifying the dog ATP8 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.9-10;

[0012] A primer pair for amplifying porcine COXI gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.11-12;

[0013] A primer pair for amplifying horse ATP6 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO. 13-14;

[0014] A primer pair for amplifying sheep NADH4 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.15-16;

[0015] A primer pair for amplifying goose 16S rRNA gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.17-18;

[0016] A primer pair for amplifying the mouse NADH4 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO. 19-20;

[0017] A primer pair for amplifying duck NADH6 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.21-22;

[0018] The primer pair for amplifying cat NADH2 gene includes an upstream primer and a downstream primer as shown in SEQ ID NO.23-24.

[0019] Furthermore, the upstream primers in the primer set are all labeled with fluorescent dyes.

[0020] Furthermore, the upstream primers for amplifying the goat D-LOOP gene, the cattle 16S rRNA gene, the donkey NADH2 gene, the chicken CYTB gene, the dog ATP8 gene and the pig COXI gene are all labeled with FAM fluorescent dye; the upstream primers for amplifying the horse ATP6 gene, the sheep NADH4 gene, the goose 16S rRNA gene, the mouse NADH4 gene, the duck NADH6 gene and the cat NADH2 gene are all labeled with HEX fluorescent dye.

[0021] The present invention also provides the use of the primer set in preparing a kit for simultaneously identifying 12 kinds of animal-derived components.

[0022] The present invention also provides a kit for simultaneously identifying 12 kinds of animal-derived components, comprising the above primer set.

[0023] Optionally, the kit further comprises 2×Multiplex PCR master Mix, 5×Q-Solution and water.

[0024] The present invention also provides a method for species identification of 12 kinds of animal-derived ingredients, comprising the following steps:

[0025] (1) Extracting DNA from the sample to be tested;

[0026] (2) performing multiple PCR amplification on the DNA using the above kit to obtain an amplified product;

[0027] (3) Performing typing detection on the amplified product and determining the species origin of the sample to be tested based on the detection result.

[0028] Optionally, the reaction system for the multiplex PCR amplification is: 2×Multiplex PCR master Mix 7.5 μL, 5×Q-Solution 1.5 μL, primer mixture 1 μL, DNA 1 μL, and water is added to 15 μL.

[0029] Optionally, in the reaction system, the final concentration of the primer pair for amplifying the goat D-LOOP gene is 0.26 μM; the final concentration of the primer pair for amplifying the cattle 16S rRNA gene is 0.5 μM; the final concentration of the primer pair for amplifying the donkey NADH2 gene is 3 μM; the final concentration of the primer pair for amplifying the chicken CYTB gene is 0.4 μM; the final concentration of the primer pair for amplifying the dog ATP8 gene is 0.6 μM; the final concentration of the primer pair for amplifying the pig COXI gene is 0.3 μM; the final concentration of the primer pair for amplifying the horse ATP6 gene is 1.6 μM; the final concentration of the primer pair for amplifying the sheep NADH4 gene is 0.3 μM; the final concentration of the primer pair for amplifying the goose 16S The final concentration of the primer pair for amplifying the rRNA gene is 0.3 μM; the final concentration of the primer pair for amplifying the mouse NADH4 gene is 0.3 μM; the final concentration of the primer pair for amplifying the duck NADH6 gene is 0.3 μM; and the final concentration of the primer pair for amplifying the cat NADH2 gene is 1.3 μM.

[0030] Optionally, the reaction procedure of the multiplex PCR amplification is: pre-denaturation at 95°C for 15 min; denaturation at 94°C for 30 s, annealing at 58°C for 90 s, extension at 72°C for 90 s, for a total of 30 cycles; final extension at 60°C for 60 min; and insulation at 4°C.

[0031] Optionally, before the typing test, the method further includes mixing the amplification product with a molecular weight internal standard and formamide; further optionally, the volume ratio of the amplification product, the molecular weight internal standard and formamide is 1:0.5:8.5.

[0032] The present invention also provides an application of the primer set, the kit or the detection method in forensic animal-derived specimen species identification or meat adulteration detection.

[0033] The present invention discloses the following technical effects:

[0034] The present invention utilizes species-specific fluorescent multiplex PCR combined with capillary electrophoresis technology to simultaneously identify biological samples of 12 common animals (goats, cattle, donkeys, chickens, dogs, pigs, horses, sheep, geese, mice, ducks and cats) at one time, amplify multiple mitochondrial DNA target sequences at the same time, and complete the identification process in the same reaction system. The overall detection time is within 3 hours, which greatly saves time and improves detection efficiency. The target fragments amplified by the present invention are all within 300bp, which can significantly improve the PCR amplification success rate of easily degradable samples and heat-processed meat products.

[0035] The DNA extraction, PCR amplification and capillary electrophoresis required for the species identification detection method established by the present invention are all standard procedures that can be performed by most forensic genetics laboratories, and can be shared by different laboratories, with a wide range of applications and strong operability. The species identification method established by the present invention is simple, rapid, highly sensitive, highly specific, and can detect a large number of species. It can provide a reliable and convenient detection method for forensic medicine and animal food identification, meet the detection needs of actual cases, and is of great significance for the rapid identification of common animal species in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, 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.

[0037] Figure 1 The genotyping patterns of the amplified products of the single DNA samples of 12 animals in Example 2;

[0038] Figure 2 is the typing pattern of the amplified product of the standard control DNA sample in Example 2;

[0039] Figure 3 The typing patterns of rabbit, frog and fish in the species-specific study in Example 3;

[0040] Figure 4 The typing patterns of pigeons, monkeys, and humans in the species-specific study in Example 3;

[0041] Figure 5 The typing pattern of the snakes used in the species-specific study in Example 3;

[0042] Figure 6 It is the detection result figure of sensitivity in Example 3;

[0043] Figure 7 This is a graph showing the test results of different mixing ratios of the mixed samples in Example 3. DETAILED DESCRIPTION

[0044] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] The invention provides a primer set for simultaneously identifying 12 kinds of animal-derived components based on mitochondrial genes, wherein the primer set comprises: a primer pair for amplifying goat D-LOOP gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.1-2; a primer pair for amplifying cattle 16S rRNA gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.3-4; a primer pair for amplifying donkey NADH2 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.5-6; a primer pair for amplifying chicken CYTB gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.7-8; a primer pair for amplifying dog ATP8 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.9-10; a primer pair for amplifying pig COXI gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.11-12; a primer pair for amplifying horse ATP6 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO. The invention relates to an upstream primer and a downstream primer as shown in SEQ ID NO.13-14 for amplifying the sheep NADH4 gene, comprising the upstream primer and the downstream primer as shown in SEQ ID NO.15-16; a primer pair for amplifying the goose 16S rRNA gene, comprising the upstream primer and the downstream primer as shown in SEQ ID NO.17-18; a primer pair for amplifying the mouse NADH4 gene, comprising the upstream primer and the downstream primer as shown in SEQ ID NO.19-20; a primer pair for amplifying the duck NADH6 gene, comprising the upstream primer and the downstream primer as shown in SEQ ID NO.21-22; a primer pair for amplifying the cat NADH2 gene, comprising the upstream primer and the downstream primer as shown in SEQ ID NO.23-24.

[0050] In some embodiments of the present invention, the upstream primers for amplifying goat D-LOOP gene, cattle 16S rRNA gene, donkey NADH2 gene, chicken CYTB gene, dog ATP8 gene and pig COXI gene are labeled with FAM fluorescent dye; the upstream primers for amplifying horse ATP6 gene, sheep NADH4 gene, goose 16S rRNA gene, mouse NADH4 gene, duck NADH6 gene and cat NADH2 gene are labeled with HEX fluorescent dye.

[0051] The present invention also provides a kit for simultaneously identifying 12 kinds of animal-derived components, comprising the above primer set.

[0052] In some embodiments of the present invention, the kit further comprises 2×Multiplex PCR master Mix, 5×Q-Solution and water.

[0053] The present invention also provides a detection method of the above kit, comprising the following steps:

[0054] (1) Extracting DNA from the sample to be tested;

[0055] (2) performing multiple PCR amplification on the DNA using the above kit to obtain an amplified product;

[0056] (3) Performing typing detection on the amplified product and determining the species origin of the sample to be tested based on the detection result.

[0057] In some embodiments of the present invention, the types of samples to be tested include but are not limited to blood, oral swabs, muscles, hair, and egg liquid.

[0058] In some embodiments of the present invention, the reaction system of the multiplex PCR amplification is: 2×Multiplex PCRmaster Mix 7.5 μL, 5×Q-Solution 1.5 μL, primer mixture 1 μL, DNA 1 μL, and water is added to 15 μL.

[0059] In some embodiments of the present invention, in the reaction system, the final concentration of the primer pair for amplifying the goat D-LOOP gene is 0.26 μM; the final concentration of the primer pair for amplifying the bovine 16S The final concentration of the primer pair for amplifying the rRNA gene is 0.5μM; the final concentration of the primer pair for amplifying the donkey NADH2 gene is 3μM; the final concentration of the primer pair for amplifying the chicken CYTB gene is 0.4μM; the final concentration of the primer pair for amplifying the dog ATP8 gene is 0.6μM; the final concentration of the primer pair for amplifying the pig COXI gene is 0.3μM; the final concentration of the primer pair for amplifying the horse ATP6 gene is 1.6μM; the final concentration of the primer pair for amplifying the sheep NADH4 gene is 0.3μM; the final concentration of the primer pair for amplifying the goose 16SrRNA gene is 0.3μM; the final concentration of the primer pair for amplifying the mouse NADH4 gene is 0.3μM; the final concentration of the primer pair for amplifying the duck NADH6 gene is 0.3μM; the final concentration of the primer pair for amplifying the cat NADH2 gene is 1.3μM.

[0060] In some embodiments of the present invention, the reaction procedure of the multiplex PCR amplification is: pre-denaturation at 95°C for 15 min; denaturation at 94°C for 30 s, annealing at 58°C for 90 s, extension at 72°C for 90 s, for a total of 30 cycles; final extension at 60°C for 60 min; and insulation at 4°C.

[0061] In some embodiments of the present invention, the multiplex PCR amplification is performed using a thermal cycler; wherein the thermal cycler can be selected from ABI 9700, ABI 9600, ABI2720, Bio-Rad iCycler or Bio-Rad C1000.

[0062] In some embodiments of the present invention, before the typing test, the step of mixing the amplified product with a molecular weight internal standard and formamide is also included; in some specific embodiments of the present invention, the volume ratio of the amplified product, the molecular weight internal standard and formamide is 1:0.5:8.5; wherein the molecular weight internal standard is T-500 and is labeled with ORG fluorescence, and the fluorescence correction is 5-Dye Matrix.

[0063] In some embodiments of the present invention, the typing detection instrument is a genetic analyzer; wherein the genetic analyzer can be selected from 3100 series, 3130 series or 3500 series genetic analyzers.

[0064] The present invention also provides an application of the primer set, the kit or the detection method in forensic animal-derived specimen species identification or meat adulteration detection.

[0065] Example 1

[0066] This example establishes a multiplex fluorescence PCR detection method that can simultaneously identify 12 common animal species, including the following steps:

[0067] 1. Screening of mitochondrial-specific DNA sequences of 12 common animals:

[0068] The mitochondrial genome reference sequences of goat (NC_005044.2), cattle (NC_006853.1), donkey (NC_001788.1), chicken (NC_053523.1), dog (NC_002008.4), pig (NC_000845.1), horse (NC_001640.1), sheep (NC_001941.1), goose (NC_011196.1), mouse (NC_005089.1), duck (NC_009684.1) and cat (NC_001700.1) were downloaded from the GenBank database. Based on the comparative genomics method, the online BLAST tool was used to perform similarity analysis on the reference sequences of the target species and non-target species, so as to screen out DNA sequences with intraspecies conservation and interspecies specificity. The specific screening steps are as follows: First, compare the mitochondrial DNA sequence of the target species with the mitochondrial DNA sequence of the non-target species, and select and retain the sequence fragments with E-value <10-5 and Percent Identity <85% as candidate sequences. For species of the same family (such as ducks and geese both belong to the Anatidae family), select the sequence fragments with E-value <10-5 and Percent Identity <90%. The inclusion criteria for species-specific DNA sequences are: ① The target sequence has sufficient base differences between different species; ② The target sequence has no base length differences within the same species distributed in different geographical regions; ③ The database has comprehensive information about the target sequence, and information on different species of the sequence can be found; ④ The target sequence length is greater than 500bp, which is convenient for primer design. The mitochondrial gene corresponding to the selected species-specific DNA sequence is marked according to the gene annotation information in GenBank. Finally, the optimal mitochondrial genes of 12 species were selected for the development of the kit, including goat D-LOOP gene, cattle 16S rRNA gene, donkey NADH2 gene, chicken CYTB gene, dog ATP8 gene, pig COXI gene, horse ATP6 gene, sheep NADH4 gene, goose 16S rRNA gene, mouse NADH4 gene, duck NADH6 gene and cat NADH2 gene.

[0069] 2. Design and effectiveness study of specific primers for 12 common animal species:

[0070] The sequences were analyzed and screened using the Ensembl database and the NCBI database, and regions with high sequence conservation and fewer SNP sites were selected. Primer Premier 5.0 software and the Primer-BLAST tool in NCBI were used to design primers that can match the selected sequences. Highly specific amplification in the target species was ensured, providing a reliable basis for subsequent experimental work. The species specificity of the primers was evaluated using the Primer-BLAST function of NCBI, and the Multiplex Manager software was used to evaluate whether there was mutual interference between multiple pairs of primers. The primers obtained were used in MFEprimer 3.1 to simulate PCR reactions on the corresponding species DNA, and the amplification between the primers and the template was analyzed, aiming to obtain species-specific amplification primers, which are expected to be applied to the construction of multiplex PCR systems.

[0071] 3. Design of fluorescent multiplex PCR amplification system:

[0072] By performing agarose gel electrophoresis on the specific primers of the 12 target species, the primers with clear, bright, and tail-free amplification product bands, amplification product lengths within the expected range, and DNA sequences obtained after bidirectional Sanger sequencing of the amplification products were selected as primers for the target species for subsequent experiments. The candidate species-specific primers were divided into two groups according to the length of the amplification product fragments. The first group was goat, cattle, donkey, chicken, dog, and pig, and the 5' end of the upstream primers was labeled with FAM fluorescent dye; the second group was horse, sheep, goose, mouse, duck, and cat, and the 5' end of the upstream primers was labeled with HEX fluorescent dye.

[0073] The mitochondrial genes of the 12 species and their corresponding amplification primer sequences, amplified fragment lengths, primer final concentrations and fluorescent labels are shown in Table 1 .

[0074] Table 1 Detailed information of amplification primers in the multiplex amplification system for identification of 12 common animal species

[0075]

[0076] 4. Construction and optimization of species-specific multiplex fluorescence PCR amplification system:

[0077] The constructed multiplex fluorescence PCR amplification system was adjusted and optimized including primer ratio concentration, DNA template amount, annealing temperature, amplification cycle number and other PCR reaction conditions until the electrophoresis typing pattern of the amplified products of the composite amplification system showed that the gene loci of each species were evenly distributed, the peaks were balanced, and there was no non-specific amplification peak. The multiplex fluorescence PCR amplification system for identifying 12 common animals is shown in Table 2, where DNA needs to be extracted from 12 common animal samples, such as blood, oral swabs, muscles, hair or egg liquid.

[0078] Table 2 Fluorescence multiplex PCR amplification system for 12 common animals

[0079] System components Volume (μL) 2×MultiplexPCRmasterMix 7.5 5×Q-Solution 1.5 Deionized water 4 Primer mix 1 DNA (1ng / μL) 1 Total volume 15

[0080] The reaction system was amplified on a thermal cycler (such as ABI 9700, ABI 9600, ABI2720, Bio-Rad iCycler or Bio-Rad C1000, etc.), and the amplification reaction program was: 95°C pre-denaturation for 15 min; 94°C denaturation for 30 s, 58°C annealing for 90 s, 72°C extension for 90 s, a total of 30 cycles; 60°C final extension for 60 min; 4°C insulation. The primer mixture in Table 2 was prepared according to the final concentration of the primers in Table 1.

[0081] 5. Establishment of analytical methods and detection of amplification products:

[0082] After establishing the 5-Dye spectral calibration file including FAM, HEX, TAMRA, ROX and ORG, capillary electrophoresis detection was performed. After mixing 1 μL of PCR amplification product with 8.5 μL of deionized formamide and 0.5 μL of molecular weight internal standard (T-500), typing detection was performed using a genetic analyzer (such as 3100 series, 3130 series or 3500 series). The electrophoretic migration parameters of mitochondrial genes of various species were obtained by capillary electrophoresis, and based on this, the electrophoretic migration parameters of mitochondrial genes of various species were calculated according to GeneMapperID v3.2.1 and The format of ID-Xv1.5 software requires the writing of corresponding Panel files and Bin files, and the creation of electrophoresis analysis methods. When editing the Panel file, the 12 gene loci in the system are edited according to the Latin abbreviations of each species and the corresponding positions of the loci genes in the mitochondria, such as goat (CH-D-LOOP), cattle (BT-16S rRNA), donkey (EA-NADH2), chicken (GG-CYTB), dog (CLF-ATP8), pig (SS-COXI), horse (EC-ATP6), sheep (OA-NADH4), goose (AC-16S rRNA), mouse (MM-NADH4), duck (AP-NADH6) and cat (FC-NADH2). When editing the Bin file, edit according to the corresponding fragment size of each species in the capillary electrophoresis map. Since the target species fragment amplified by the present invention has only one genotype, in order to make the map balanced, the gene fragment size of the Bin set is set within the range of 10-20bp from the target gene fragment length. In addition, in order to more intuitively identify the electrophoresis typing pattern, the alleles are no longer named based on the fragment length. Instead, the alleles are named based on the English names of the 12 species in the system, namely goat, cattle, donkey, chicken, dog, pig, horse, sheep, goose, rat, duck and cat.

[0083] Example 2

[0084] The method established in Example 1 was used to detect 12 kinds of animal DNA samples and a standard control DNA sample. The specific operation is as follows:

[0085] Samples from 12 kinds of animals, including goats, cattle, donkeys, chickens, dogs, pigs, horses, sheep, geese, rats, ducks and cats, including blood, oral swabs, muscles, hair and egg liquid, were collected for DNA extraction and quantification.

[0086] The DNA templates of goat, cattle, donkey, chicken, dog, pig, horse, sheep, goose, mouse, duck and cat were mixed in equal proportions to prepare a standard control DNA sample, and the final concentration of the standard control DNA was 1 ng / μL.

[0087] The above 12 kinds of animal DNA samples and standard control DNA samples were amplified using the method constructed in Example 1, and the amplified products were detected by capillary electrophoresis.

[0088] The results of the DNA samples of 12 animals are as follows Figure 1 As shown in the figure, the amplification peaks corresponding to the gene loci of each species are single, and the fragment lengths are in line with expectations. The test results of the standard control DNA sample are as follows Figure 2 (The upper row from left to right are CH-D-LOOP, BT-16S rRNA, EA-NADH2, GG-CYTB, CLF-ATP8, SS-COXI, the lower row from left to right are EC-ATP6, OA-NADH4, AC-16S rRNA, MM-NADH4, AP-NADH6, FC-NADH2) shows that the gene loci of each species are evenly distributed, the peaks are balanced, and there are no nonspecific amplification peaks.

[0089] Example 3

[0090] The method established in Example 1 was forensically validated (species specificity, sensitivity, repeatability and mixture) according to the SWGDAM requirements to evaluate the accuracy, reliability and stability of the method. The specific operations are as follows:

[0091] 1. Species-specific study: DNA samples of seven species, including human, rabbit, monkey, pigeon, snake, frog and fish, were quantified to 2 ng / μL, and the DNA of the seven non-target species were detected separately using the method established in Example 1.

[0092] The results are as follows Figure 3-Figure 5 As shown, in each species detection result diagram, the upper row from left to right is CH-D-LOOP, BT-16S rRNA, EA-NADH2, GG-CYTB, CLF-ATP8, SS-COXI, and the lower row from left to right is EC-ATP6, OA-NADH4, AC-16S rRNA, MM-NADH4, AP-NADH6, FC-NADH2. It can be seen that the method of Example 1 cannot be successfully amplified in the DNA of the above 7 species, and no amplification peaks are detected in the 7 species. It shows that this method has good species specificity, non-target DNA contamination samples will not interfere with the accuracy of the test results, and can meet the species identification of complex biological samples at the crime scene in actual situations.

[0093] 2. Sensitivity study: Species with relatively low fluorescence peaks in the standard control DNA sample test results were selected for sensitivity study, and two species were selected for each color of fluorescence (FAM for goats and donkeys, HEX for sheep and ducks). The DNA of four species of goats, donkeys, sheep and ducks were diluted to 2ng / μL, 1ng / μL, 0.5ng / μL, 0.25ng / μL, 0.125ng / μL, 0.0625ng / μL and 0.03125ng / μL, and the target species DNA at the above concentrations were detected respectively using the method established in Example 1, and each concentration was tested in parallel 3 times.

[0094] The results are as follows Figure 6As shown in the figure, as the amount of DNA template decreases, the peak height of the amplified product in capillary electrophoresis gradually decreases, but the corresponding target peaks of the four species can still be detected when the DNA amount is 0.03125ng. This shows that the method has high sensitivity, and this sensitivity level can meet the needs of actual cases.

[0095] 3. Repeatability study: 12 target species DNA samples (3 samples for each species) were tested using the method established in Example 1 to analyze the fragment sizes in the genetic analysis maps of different individuals of the same species.

[0096] The results showed that the typing results obtained for each species were consistent, and the difference in the length of the amplified peak fragments did not exceed 0.5 bp, which could accurately identify the species type, indicating that this method has good repeatability.

[0097] 4. Mixed sample study: Slice the duck and beef into thin slices and place them in an oven at 75°C, remove moisture and grind into powder. Mix the ground duck meat into beef at a mass ratio of 0.5%, 1%, 5%, and 10%. The total mass of each simulated mixed sample is 100 mg, and DNA is extracted after thorough mixing. The DNA of the above different mass ratios is detected using the method established in Example 1, and each sample is tested in parallel 3 times.

[0098] The results are as follows Figure 7 As shown, the four proportions of the mixture can accurately detect duck and cattle species components. When the mixed sample ratio is as low as 0.5%, that is, when the amount of non-beef components is as low as 0.5 mg, non-beef components can still be detected in the mixture. This shows that this method can meet the requirements of meat adulteration detection in actual situations.

[0099] It can be seen from the above embodiments that the primer set, kit and method for simultaneously identifying 12 common animal species provided by the present invention can provide a reliable and convenient detection means for forensic medicine and animal food identification, meet the detection needs of actual cases, and are of great significance for the rapid identification of common species in my country.

[0100] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer set for simultaneously identifying 12 animal-derived ingredients based on mitochondrial genes, characterized in that: The primer set comprises: A primer pair for amplifying goat D-LOOP gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.1-2; A primer pair for amplifying the bovine 16S rRNA gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.3-4; A primer pair for amplifying the donkey NADH2 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.5-6; A primer pair for amplifying chicken CYTB gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.7-8; A primer pair for amplifying the dog ATP8 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.9-10; A primer pair for amplifying porcine COXI gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.11-12; A primer pair for amplifying horse ATP6 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO. 13-14; A primer pair for amplifying sheep NADH4 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.15-16; A primer pair for amplifying goose 16S rRNA gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.17-18; A primer pair for amplifying the mouse NADH4 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO. 19-20; A primer pair for amplifying duck NADH6 gene, comprising an upstream primer and a downstream primer as shown in SEQ ID NO.21-22; The primer pair for amplifying cat NADH2 gene includes an upstream primer and a downstream primer as shown in SEQ ID NO.23-24.

2. The primer set according to claim 1, characterized in that The upstream primers in the primer set are all labeled with fluorescent dyes.

3. The primer set according to claim 2, characterized in that The upstream primers for amplifying the goat D-LOOP gene, the cattle 16SrRNA gene, the donkey NADH2 gene, the chicken CYTB gene, the dog ATP8 gene and the pig COXI gene are all labeled with FAM fluorescent dye; the upstream primers for amplifying the horse ATP6 gene, the sheep NADH4 gene, the goose 16S rRNA gene, the mouse NADH4 gene, the duck NADH6 gene and the cat NADH2 gene are all labeled with HEX fluorescent dye.

4. Use of the primer set according to any one of claims 1 to 3 in preparing a kit for simultaneously identifying 12 animal-derived components.

5. A kit for simultaneously identifying 12 animal-derived ingredients, characterized in that: A primer set comprising any one of claims 1 to 3.

6. A method for species identification of 12 animal-derived ingredients, characterized in that: The steps include: (1) Extracting DNA from the sample to be tested; (2) performing multiple PCR amplification on the DNA using the kit described in claim 5 to obtain an amplified product; (3) Performing typing detection on the amplified product and determining the species origin of the sample to be tested based on the detection result.

7. The detection method according to claim 6, characterized in that: The reaction system of the multiplex PCR amplification is: 2×Multiplex PCR master Mix 7.5 μL, 5×Q-Solution 1.5 μL, primer mixture 1 μL, DNA 1 μL, and water is added to 15 μL.

8. The method according to claim 7, characterized in that In the reaction system, the final concentration of the primer pair for amplifying goat D-LOOP gene is 0.26 μM; the final concentration of the primer pair for amplifying cattle 16S rRNA gene is 0.5 μM; the final concentration of the primer pair for amplifying donkey NADH2 gene is 3 μM; the final concentration of the primer pair for amplifying chicken CYTB gene is 0.4 μM; the final concentration of the primer pair for amplifying dog ATP8 gene is 0.6 μM; the final concentration of the primer pair for amplifying pig COXI gene is 0.3 μM; the final concentration of the primer pair for amplifying horse ATP6 gene is 1.6 μM; the final concentration of the primer pair for amplifying sheep NADH4 gene is 0.3 μM; the final concentration of the primer pair for amplifying goose 16S rRNA gene is 0.3 μM; the final concentration of the primer pair for amplifying mouse NADH4 gene is 0.3 μM; the final concentration of the primer pair for amplifying duck NADH6 gene is 0.3 μM; and the final concentration of the primer pair for amplifying cat NADH2 gene is 1.3 μM.

9. The method according to claim 6, wherein the reaction procedure of the multiplex PCR amplification is: pre-denaturation at 95°C for 15 min; denaturation at 94°C for 30 s, annealing at 58°C for 90 s, extension at 72°C for 90 s, for a total of 30 cycles; final extension at 60°C for 60 min; and insulation at 4°C.

10. Use of the primer set according to any one of claims 1 to 3, the kit according to claim 5, or the method according to any one of claims 6 to 9 in forensic animal-derived specimen species identification or meat adulteration detection.

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