Primer pair combination for identifying animal species and application of primer pair combination in kit

Abstract

The invention belongs to the technical field of molecular biology, and relates to a primer pair combination for identifying animal species and application of the primer pair combination in a kit. According to the method, the identification efficiency and accuracy are remarkably improved, PCR amplification is carried out by utilizing various gene markers (such as COI, cyt b, 16S rRNA and 12S rRNA), and individual differences of different families and even in the same species can be effectively distinguished due to high conservative property and variability of the gene regions in different species. For example, primer pair sequences SEQ ID NO.1-2 and 9-10 are designed for general animal species, and SEQ ID NO.3-4 are specially optimized for bird animal species, so that the design ensures that accurate results can be obtained even for complex sample types.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular chemistry and relates to a primer pair combination for identifying animal species and an application of the primer pair combination in a kit. Background Art

[0002] In recent years, with my country's growing awareness of ecological and environmental protection, public security departments have been stepping up their crackdowns on crimes that endanger the environment and damage natural resources and the environment. The need for DNA identification of animal specimens has become particularly urgent, particularly given the significant increase in cases involving animal species identification. As the world's third-largest country by land area, my country boasts a diverse range of climate zones, from tropical and subtropical to temperate. This has not only created a rich natural landscape but also fostered exceptional biodiversity. According to the latest edition of the "List of Biological Species of China 2024," my country now has a record of 69,407 animal species, including 234 first-class and 746 second-class protected species. These rare species play an irreplaceable role in maintaining ecological balance, economic development, and the preservation of social and cultural heritage.

[0003] In order to more effectively combat crimes involving animal resources, DNA identification projects for non-human biological samples have been added to the qualification certification of testing laboratories of identification agencies of public security organs since July 2021. This move marks an important step forward for my country in animal species identification technology. At present, animal species identification mainly relies on two methods: morphological identification and molecular identification. Among them, morphological identification determines the identity of the species by observing the overall morphological characteristics of individual organisms, but it is powerless when dealing with animal tissues that have lost macroscopic morphological characteristics, which increases the complexity of case investigation. In contrast, molecular identification, especially DNA-based technology, provides more accurate and reliable solutions, but the current public security forensic DNA laboratories have insufficient practical experience and technical accumulation in this regard, and there is a lack of mature and stable commercial test kits on the market to support related work.

[0004] To address these challenges, the project team plans to build and optimize a technical system suitable for DNA species identification of animal specimens and is committed to developing corresponding DNA identification kits. This initiative aims to fill a gap in this field domestically, providing strong technical support and service guarantees to public security departments, market supervision and administration bureaus, scientific research institutions, and others, meeting their practical needs in animal protection, biosafety monitoring, and food safety regulation. In the long run, the successful implementation of this project will not only help enhance my country's scientific and technological level in animal resource conservation but will also have a positive and far-reaching impact on global biodiversity conservation. At the same time, by strengthening international cooperation and exchanges, we will promote the internationalization of relevant technologies and standards, and jointly address the increasingly severe ecological and environmental challenges worldwide. Summary of the Invention

[0005] The purpose of the present invention is to address the above-mentioned problems existing in the prior art and propose a primer pair combination for identifying animal species. It uses multiple pairs of universal primers to cover the detection of all types of animal species and is suitable for DNA identification of species from animal tissues, hair, bones, etc.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A primer pair combination for identifying animal species, wherein the primer pair combination sequence is shown as SEQ ID NO. 1-16.

[0008] In the above-mentioned primer pair combination for identifying animal species, the primer pair sequence SEQ ID NO. 1-2 is used for PCR amplification using the COI gene as the target gene;

[0009] The primer pair sequence SEQ ID NO.3-4 was used for PCR amplification with COI gene as the target gene;

[0010] Primer pair sequence SEQ ID NO.5-6 was used for PCR amplification with cyt b gene as the target gene;

[0011] The primer pair sequence SEQ ID NO.7-8 was used for PCR amplification with cyt b gene as the target gene;

[0012] Primer pair sequence SEQ ID NO.9-10 was used for PCR amplification with 16S rRNA gene as the target gene;

[0013] Primer pair sequence SEQ ID NO.11-12 was used for PCR amplification with 16S rRNA gene as the target gene;

[0014] Primer pair sequence SEQ ID NO. 13-14 was used for PCR amplification with 12S rRNA gene as the target gene;

[0015] The primer pair sequence SEQ ID NO. 15-16 was used for PCR amplification with 12S rRNA gene as the target gene.

[0016] In the above-mentioned primer pair combination for identifying animal species, the primer pair sequences SEQ ID NO. 1-2 are used to identify universal animal species;

[0017] Primer pair sequence SEQ ID NO.3-4 is used to identify avian animal species;

[0018] Primer pair sequences SEQ ID NO. 5-8 were used to identify vertebrate species;

[0019] Primer pair sequences SEQ ID NO. 9-10 are used to identify common animal species;

[0020] Primer pair sequences SEQ ID NO. 11-16 were used to identify vertebrate species.

[0021] A kit for identifying animal species, comprising the above primer pair combination.

[0022] In the above-mentioned kit for identifying animal species, the kit further includes an extraction kit, an amplification kit, a PCR product purification kit, a sequencing kit, and a sequencing product purification kit.

[0023] Preferably, the extraction kit comprises lysis solution, proteinase K, magnetic beads, binding solution, washing solution I, washing solution II, washing solution III, and elution solution.

[0024] The lysis buffer includes buffer, salt ions, detergents, and protease inhibitors.

[0025] The binding solution includes buffer, salt ions, and isopropanol.

[0026] Washing solutions I, II, and III include buffer, salt ions, and ethanol.

[0027] The eluent consisted primarily of nuclease-free water.

[0028] The amplification kit includes Master, Buffer, primers, and nuclease-free water.

[0029] The Master includes a buffer solution containing MgCl2.

[0030] Buffer includes a mixture of enzymes.

[0031] The PCR product purification kit contains magnetic beads, binding solution, washing solution I, washing solution II, washing solution III, and elution solution.

[0032] The binding solution includes buffer, salt ions, and isopropanol.

[0033] Washing solutions I, II, and III include buffer, salt ions, and ethanol.

[0034] Elution buffer consisted of nuclease-free water.

[0035] The sequencing kit contains Master, Buffer, primers, and nuclease-free water.

[0036] The Master includes a buffer containing MgCl2.

[0037] Buffer includes an enzyme mixture.

[0038] The sequencing product purification kit contains Purification Solution and Resin.

[0039] Purification Solution includes buffer and salt ions.

[0040] Resin is mainly composed of resin.

[0041] A method for identifying an animal species, comprising the following steps:

[0042] S1. Extract DNA from animal samples to be identified;

[0043] S2. Using the DNA from step S1 as a template, perform PCR amplification using the above primer pair combination to obtain a PCR product;

[0044] S3. The PCR products were purified and sequenced and compared with the published gene sequences of animal species.

[0045] In the above-mentioned method for identifying animal species, the gene sequence comparison results: when the gene sequence identity is ≥99%, it is determined to be the same species; when the identity is ≥90% but <99%, it is determined to be species of the same family or genus.

[0046] As a preference, when the consistency is ≥99%, the source of the sample tested is determined to be the species with the highest consistency;

[0047] When the consistency is ≥90% but <99%, the sample is determined to be from the XX family or XX genus of the species with the highest consistency; or the mtDNA barcode of a different gene segment is replaced and retested to determine the species to which the sample belongs;

[0048] When the consistency is less than 90%, the cause should be analyzed, the experiment should be repeated, and the mtDNA barcode of different gene fragments should be replaced and retested.

[0049] If the consistency is still less than 90% after multiple tests, this method is not suitable for the sample and should be supplemented by other identification methods;

[0050] When comparing the base sequence with the database, it may be found that the match and homology with several closely related species are exactly the same. In this case, the mtDNA barcodes of other gene fragments should be replaced for verification.

[0051] In the above-mentioned method for identifying animal species, the PCR amplification reaction system is 20 μL, that is, 10-20 μL DNase / RNase Free H2O, 3-5 μL Species Identification Master Mix, 2.5-2.8 μL Species Identification Buffer, 0.1-0.3 μL of each primer (200-250 μM), and 1-13 μL DNA (1-30 ng / μL) are added.

[0052] Preferably, the Species Identification Master Mix comprises: 15wt% MgCl2, 2.5wt% Tris-HCl, 82.5wt% pure water; the Species Identification Buffer comprises: 10wt% dNTPs, 5wt% DNA Polymerase, 85wt% pure water.

[0053] In the above-mentioned method for identifying animal species, the PCR reaction conditions are: pre-denaturation at 95-98°C for 0.5-1.5 minutes; denaturation at 95-98°C for 5-15 seconds, annealing at 45-55°C for 3-8 seconds, extension at 55-65°C for 2-5 minutes, and 30-40 cycles; and final extension at 3-5°C.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] 1. The present invention proposes a primer pair combination for identifying animal species. By adopting multiple pairs of universal primers, it covers the detection needs of all kinds of animal species and is particularly suitable for DNA identification of species from animal tissues, hair, bones, etc.

[0056] 2. This invention significantly improves identification efficiency and accuracy by utilizing multiple gene markers (such as COI, cyt b, 16S rRNA, and 12S rRNA) for PCR amplification. These gene regions, due to their high conservation and variability across species, can effectively distinguish between individuals within different families and even within the same species. For example, primer pairs SEQ ID NOs. 1-2 and 9-10 are designed for common animal species, while SEQ ID NOs. 3-4 are specifically optimized for avian species. This design ensures accurate results even with complex sample types.

[0057] 3. The present technology is not limited to the complete morphological features of an organism and is particularly suitable for processing animal tissues that have lost their macroscopic morphological features, thus resolving a long-standing technical challenge in forensic identification. Furthermore, because the present invention, based on DNA barcoding technology, relies on genetic information rather than phenotypic characteristics, it also performs well across developmental stages or in situations with significant morphological variation, providing a more reliable and accurate method for species identification.

[0058] 4. This invention not only proposes an innovative set of primer pairs but also develops a comprehensive kit, including extraction, amplification, PCR product purification, sequencing, and detection reagents for the entire process of sequencing product purification. This comprehensive solution greatly simplifies the operational process, improves laboratory efficiency, and reduces the risk of errors caused by manual operation. It also provides strong technical support and service guarantees for relevant institutions such as scientific research institutions, public security departments, and market supervision and administration bureaus, meeting their urgent needs in animal protection, biosafety monitoring, and food safety supervision.

[0059] 5. By establishing clear gene sequence alignment criteria (gene sequence identity ≥99% is considered the same species, and identity ≥90% but <99% is considered the same family or genus), this invention further enhances the objectivity and scientific nature of identification results. This method not only enables rapid and accurate identification of unknown species, but also contributes to the establishment of a more systematic and standardized animal species identification system, promoting the healthy development and technological advancement of related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The comparison results are shown for 1 ng / μL bovine DNA as the template;

[0061] Figure 2 The comparison results are shown for 5 ng / μL bovine DNA as the template;

[0062] Figure 3 The comparison results are shown for 7.5 ng / μL bovine DNA as the template;

[0063] Figure 4 The comparison results are shown for 10 ng / μL bovine DNA as the template;

[0064] Figure 5 The comparison results are shown for 15 ng / μL bovine DNA as the template;

[0065] Figure 6 This is the comparison result using 20 ng / μL bovine DNA as template;

[0066] Figure 7The comparison results are shown for 30 ng / μL bovine DNA as the template;

[0067] Figure 8 The comparison results are shown for 10 ng / μL bovine DNA as the template;

[0068] Figure 9 The comparison results are shown using 10 ng / μL turtledove DNA as a template;

[0069] Figure 10 The comparison results are shown using 10 ng / μL of goitered gazelle DNA as a template;

[0070] Figure 11 The comparison results are shown for 10 ng / μL Eurasian wild boar DNA as a template;

[0071] Figure 12 The comparison results are shown for 10 ng / μL small yellow croaker DNA as template;

[0072] Figure 13 The comparison results are shown for 10 ng / μL domestic cat DNA as a template;

[0073] Figure 14 This is the alignment result using 10 ng / μL dog DNA as the template;

[0074] Figure 15 The comparison results are shown for 10 ng / μL pigeon DNA as template;

[0075] Figure 16 The comparison results are shown for 10 ng / μL sheep DNA as template;

[0076] Figure 17 The comparison results are shown for 10 ng / μL leopard cat DNA as a template;

[0077] Figure 18 The comparison results are shown using 10 ng / μL Turtle Dove DNA as the template.

[0078] Figure 19 The comparison results are shown for the red junglefowl DNA with a concentration of 10 ng / μL as the template.

[0079] Figure 20 The comparison results are shown using 10 ng / μL of dove bone DNA as a template;

[0080] Figure 21 The comparison results are shown using 10 ng / μL of dove tissue DNA as a template.

[0081] Figure 22 The comparison results are shown using 10 ng / μL turtledove hair DNA as a template; DETAILED DESCRIPTION

[0082] Below by specific embodiment and accompanying drawing, technical scheme of the present invention is further described explanation, it should be understood that specific embodiment described herein is only for helping to understand the present invention, is not used for specific limitation of the present invention.And accompanying drawing used herein, is only for better illustrating the disclosure of the present invention, does not have limiting effect on protection scope.If no special instructions, the raw materials adopted in the embodiment of the present invention are all raw materials commonly used in this area, and the method adopted in the embodiment is all conventional method in this area.

[0083] Detection time: 1-3 days

[0084] Sample types: including animal tissues, bones, hair, products, remains, body fluids, metabolites, etc.

[0085] Table 1: Primer sequences

[0086]

[0087] S1. Extraction:

[0088] (1) Lysis / bone powder incubation specimen types: tissue, bloodstains, hair;

[0089] Place an appropriate amount of sample into a 1.5 mL centrifuge tube, add 400 μL of lysis buffer and 10 μL of proteinase K, and mix well.

[0090] Lysis solution material content: 45 wt% of guanidine hydrochloride, 4 wt% of 1 M Tris-HCl solution, 1 wt% of 0.5 M EDTA solution, and 50 wt% of pure water.

[0091] Heat treatment of the mixed solution: 800 rpm, 56 ° C for 30 min (the time can be appropriately extended to improve the cracking effect).

[0092] After heat treatment, centrifuge at 12000 rpm for 3 minutes, take the supernatant and transfer it into a new 1.5 mL centrifuge tube and mark it.

[0093] Sample type: bones, teeth

[0094] Place an appropriate amount of bone powder (approximately 0.5g-2g) into a suitable centrifuge tube. Add the reagents at a ratio of 1.5mL of bone powder incubation solution and 75μL (20mg / mL) of proteinase K per 1g of bone powder. Mix thoroughly and incubate at 51°C for 24-48 hours. Shake vigorously at least 5-6 times during incubation, and add an equal amount of proteinase K approximately 12 hours into the incubation. After incubation, centrifuge at 12,000 rpm for 5 minutes. Transfer the supernatant to a new 1.5mL centrifuge tube and label it.

[0095] (2) Combination

[0096] The binding liquid contains the following substances: 52 wt% of guanidine hydrochloride, 30 wt% of isopropyl alcohol, and 18 wt% of pure water.

[0097] Add 21 μL of mixed magnetic beads and 350 μL of binding buffer to the supernatant of the lysis or incubation. Vortex to mix thoroughly. Incubate at room temperature for 10 minutes, vortexing every minute to ensure the beads remain suspended. Place the centrifuge tube on a magnetic stand and invert the stand 3-4 times to perform magnetic separation. Discard the waste liquid by aspiration.

[0098] (3) Washing

[0099] ① Add 450 μL of washing solution I and vortex for 1 minute; place the centrifuge tube on the magnetic rack and invert it together with the magnetic rack 3-4 times for magnetic separation, and discard the waste liquid.

[0100] The substance content of the washing liquid I is: 35 wt% of guanidine hydrochloride, 50 wt% of isopropyl alcohol, and 15 wt% of pure water.

[0101] ② Add 450 μL of washing solution II and vortex for 1 minute; place the centrifuge tube on the magnetic rack, invert it together with the magnetic rack 3-4 times for magnetic separation, and discard the waste liquid.

[0102] The substance content of washing liquid II is: anhydrous ethanol 72wt%, pure water 28wt%.

[0103] ③ Add 450 μL of Wash III and vortex for 1 minute; place the centrifuge tube on the magnetic rack, invert it together with the magnetic rack 3-4 times for magnetic separation, and discard the waste liquid.

[0104] The substance content of the washing liquid III is: 63 wt% of anhydrous ethanol and 37 wt% of pure water.

[0105] (4) Elution

[0106] Uncap the centrifuge tube and allow it to stand at room temperature for 2 minutes to allow the beads to dry. Add 50 μL of elution buffer and vortex to mix thoroughly. Incubate the mixture at 800 rpm at 65°C for 10 minutes. Centrifuge briefly and place the tube on a magnetic stand for magnetic separation. Carefully aspirate the supernatant and transfer it to a new 1.5 mL centrifuge tube to obtain the purified DNA sample. (Recommended storage temperature: 2°C to 8°C for short-term storage and -15°C to -25°C for long-term storage.)

[0107] Eluent substance content: pure water 100wt%.

[0108] S2. Amplification

[0109] Table 2: Reaction system preparation

[0110]

[0111] Note: The recommended DNA input amount is >30 ng. The DNA loading amount can be adjusted according to the DNA concentration.

[0112] Table 3: Amplification parameter settings

[0113]

[0114] S3. PCR product purification

[0115] (1) Combined:

[0116] The binding liquid contains the following substances: 56 wt % of guanidine hydrochloride, 28 wt % of isopropyl alcohol, and 16 wt % of pure water.

[0117] Transfer 20 μL of PCR product to a centrifuge tube, add 200 μL of binding buffer and 15 μL of magnetic beads, and mix thoroughly to fully disperse the beads. Vortex to mix thoroughly and place on a mixer for 10 minutes. Place the centrifuge tube on a magnetic stand for 2 minutes, invert the stand five times, and let it sit for 1 minute to allow the beads to completely absorb. Use a pipette to remove as much liquid as possible without touching the beads.

[0118] (2) Washing:

[0119] ① Remove the centrifuge tube from the magnetic rack, add 500 μL of Wash Buffer I, and vortex 5-10 times to fully disperse the magnetic beads. Then, place the centrifuge tube on the magnetic rack for 2 minutes until the magnetic beads are completely absorbed. Use a pipette to discard all liquid, avoiding contact with the magnetic beads.

[0120] The substance content of washing solution I is: 30 wt% of guanidine hydrochloride, 50 wt% of isopropyl alcohol, 1 wt% of 0.5 M EDTA solution, and 19 wt% of pure water.

[0121] ②Add 500 μL of washing solution II and repeat step 4.

[0122] The substance content of washing liquid II is: anhydrous ethanol 72wt%, pure water 28wt%.

[0123] ③Add 500 μL of washing solution III and repeat step 4.

[0124] The substance content of the washing liquid III is: 58 wt% of anhydrous ethanol and 42 wt% of pure water.

[0125] ④ Briefly centrifuge, place the centrifuge tube on a magnetic rack to absorb the residual liquid, and dry it at room temperature (the specific time is based on the principle of complete drying of the magnetic beads).

[0126] (3) Elution: Add 50 μL of elution buffer, shake and mix for 5 minutes. After a brief centrifugation, place the centrifuge tube on a magnetic stand for 2 minutes. Use a pipette to transfer the supernatant to another clean centrifuge tube. Store the purified DNA at -20°C.

[0127] Eluent substance content: pure water 100%.

[0128] S4, sequencing (amplification)

[0129] Table 4: Reaction system preparation

[0130]

[0131] Note: 1) The recommended DNA input amount is >10 ng. The DNA loading amount can be adjusted according to the DNA concentration.

[0132] Primer refers to a single upstream or downstream primer.

[0133] Table 5: Amplification parameter settings

[0134]

[0135] Note: 1) The status of amplification instruments in different laboratories may vary. The number of cycles can be adjusted according to the laboratory instrument status and actual needs.

[0136] 2) If the DNA concentration is low, the number of cycles can be changed to 40, and the extension time can be changed from 4 min to 2 min.

[0137] S5. Sequencing product purification

[0138] Table 6: Preparation of premix

[0139] Reagent components volume Sequencing products 10 μL Purification Solution 45 μL Resin 10 μL

[0140] After the premix is ​​prepared, shake it on a shaker for 30 minutes, centrifuge it at 3500 rpm for 2 minutes, and take 10 μL of the supernatant for the test.

[0141] Comparison of test results: The original results of the sequencer test are exported to the analysis software and analyzed. After the base sequence is obtained, the sequence consistency is compared using the NCBI blast function. Sequence consistency: The gene sequence consistency of the sample sequence is compared with the database sequence. The gene sequence consistency should be at least ≥90%, including:

[0142] 1. When the consistency is ≥99%, the sample is determined to be from the species with the highest consistency. 2. When the consistency is ≥90% but <99%, the sample is determined to be from the XX family or XX genus of the species with the highest consistency. Alternatively, the mtDNA barcode of a different gene segment should be replaced and retested to determine the species to which the sample belongs. 3. When the consistency is <90%, the cause should be analyzed, the experiment should be repeated, and the mtDNA barcode of a different gene segment should be replaced and retested. 4. If the consistency is still <90% after multiple tests, this method is not applicable to the sample and should be supplemented with other identification methods. 5) When the base sequence is compared with the database, it may be found that the match and homology with several closely related species are exactly the same. In this case, the mtDNA barcode of other gene segments should be replaced for verification.

[0143] Example 1:

[0144] 1 ng / μL, 5 ng / μL, 7.5 ng / μL, 10 ng / μL, 15 ng / μL, 20 ng / μL, and 30 ng / μL of bovine DNA were used as templates, respectively.

[0145] Table 7: Amplification system

[0146]

[0147] Table 8: Amplification Parameters

[0148]

[0149] The subsequent steps, including PCR product purification, cycle sequencing, and sequencing product purification, maintained the same conditions.

[0150] from Figure 1-7 The results showed that at 7.5 ng, the consistency was 91.9% for domestic cattle; at 10 ng, the consistency was 100% for domestic cattle; at 10 ng, the consistency was 100% for domestic cattle; at 15 ng, the consistency was 100% for domestic cattle; at 20 ng, the consistency was 100% for domestic cattle; and at 30 ng, the consistency was 100% for domestic cattle. This indicates that even with a template amount of 10 ng, the sequencing results of this system are accurate, with a consistency of >99% in NCBI.

[0151] Example 2:

[0152] 10 ng of DNA extracts from cattle, mountain dove, goitered gazelle, Eurasian wild boar, small yellow croaker, domestic cat, rock pigeon, sheep, leopard cat, spotted dove, and red jungle fowl were used as templates respectively.

[0153] Table 9: Amplification system

[0154]

[0155] Table 10: Amplification Parameters

[0156]

[0157] from Figure 8-19 The experimental results show that: the consistency of domestic cattle is 100%, the consistency of mountain dove is 99.38%, the consistency of goitered gazelle is 100%, the consistency of Eurasian wild boar is 99.79%, the consistency of small yellow croaker is 100%, the consistency of domestic cat is 100%, the consistency of dog is 100%, the consistency of rock pigeon is 99.34%, the consistency of sheep is 99.73%, the consistency of leopard cat is 99.12%, the consistency of spotted dove is 100%, and the consistency of red jungle fowl is 100%.

[0158] Example 3:

[0159] 10 ng of DNA extracts from the bones, tissues and hair of the mountain dove were used as templates respectively.

[0160] Table 11: Amplification system

[0161]

[0162] Table 12: Amplification Parameters

[0163]

[0164] from Figure 20-22 The experimental results show that the comparison results of different types of samples of the same sample are the same, and the consistency is >99%.

[0165] In summary, the primer pair combination for identifying animal species proposed in the present invention covers the detection needs of all types of animal species by adopting multiple pairs of universal primers, and is particularly suitable for DNA identification of species from animal tissues, hair, bones, etc.

[0166] The parts of the embodiment herein that are not exhaustive of the midpoint values ​​of the technical scope claimed for protection by the present invention and the new technical solutions formed by equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope claimed for protection by the present invention; at the same time, in all the embodiments listed or not listed in the solutions of the present invention, each parameter in the same embodiment merely represents an example of its technical solution (i.e., a feasible solution), and there is no strict coordination and limitation relationship between the parameters, wherein the parameters can be replaced with each other without violating the axioms and the claims of the present invention, unless otherwise stated.

[0167] The technical means disclosed in the solutions of the present invention are not limited to the technical means disclosed in the above technical means, but also include technical solutions composed of any combination of the above technical features. The above is a specific embodiment of the present invention. It should be noted that for those skilled in the art, various improvements and modifications can be made without departing from the principles of the present invention, and such improvements and modifications are also considered to be within the scope of protection of the present invention.

[0168] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.

Claims

1. A primer pair combination for identifying animal species, characterized in that: The primer pair combination sequences are shown in SEQ ID NO. 1-16.

2. A primer pair combination for identifying animal species according to claim 1, characterized in that: The primer pair sequence SEQ ID NO.1-2 was used for PCR amplification with COI gene as the target gene; The primer pair sequence SEQ ID NO.3-4 was used for PCR amplification with COI gene as the target gene; Primer pair sequence SEQ ID NO.5-6 was used for PCR amplification with cyt b gene as the target gene; The primer pair sequence SEQ ID NO.7-8 was used for PCR amplification with cyt b gene as the target gene; Primer pair sequence SEQ ID NO.9-10 was used for PCR amplification with 16S rRNA gene as the target gene; Primer pair sequence SEQ ID NO.11-12 was used for PCR amplification with 16S rRNA gene as the target gene; Primer pair sequence SEQ ID NO. 13-14 was used for PCR amplification with 12S rRNA gene as the target gene; The primer pair sequence SEQ ID NO. 15-16 was used for PCR amplification with 12S rRNA gene as the target gene.

3. A primer pair combination for identifying animal species according to claim 1 or 2, characterized in that: Primer pair sequences SEQ ID NO. 1-2 are used to identify common animal species; Primer pair sequence SEQ ID NO.3-4 is used to identify avian animal species; Primer pair sequences SEQ ID NO. 5-8 were used to identify vertebrate species; Primer pair sequences SEQ ID NO. 9-10 are used to identify common animal species; Primer pair sequences SEQ ID NO. 11-16 were used to identify vertebrate species.

4. A kit for identifying animal species, characterized in that The kit comprises the primer pair combination according to claim 1.

5. A kit for identifying animal species according to claim 4, characterized in that: The kit also includes an extraction kit, an amplification kit, a PCR product purification kit, a sequencing kit, and a sequencing product purification kit.

6. A method for identifying animal species, characterized in that: The method comprises the following steps: S1. Extract DNA from animal samples to be identified; S2. Using the DNA from step S1 as a template, perform PCR amplification using the primer pair combination of claim 1 to obtain a PCR product; S3. The PCR products were purified and sequenced and compared with the published gene sequences of animal species.

7. The method for identifying animal species according to claim 6, wherein: Gene sequence comparison results: When the gene sequence identity is ≥99%, it is determined to be the same species; when the identity is ≥90% but <99%, it is determined to be species of the same family or genus.

8. The method for identifying animal species according to claim 6, wherein: The PCR amplification reaction system is 20 μL, that is, add 10-20 μL DNase / RNase Free H2O, 3-5 μL Species Identification Master Mix, 2.5-2.8 μL Species Identification Buffer, 0.1-0.3 μL of each primer (200-250 μM), and 1-13 μL DNA (1-30 ng / μL).

9. The method for identifying animal species according to claim 6, wherein: The PCR reaction conditions were as follows: pre-denaturation at 94-98°C for 0.5-1.5 min; denaturation at 94-98°C for 5-15 s, annealing at 45-55°C for 3-8 s, extension at 55-75°C for 2-5 min, and 30-40 cycles; and final extension at 3-5°C.

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