Primer and kit for specifically identifying sheepskin and identification method of sheepskin
By designing the specific primer KLHL17 and combining PCR amplification and agarose gel electrophoresis technology, the problem of sheepskin identification was solved, and a fast, simple and accurate identification effect was achieved, reducing equipment requirements and improving identification efficiency.
Patent Information
- Application Number
- CN202510545516.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-07-25
AI Technical Summary
The prior art is difficult to quickly, easily and accurately identify sheepskin, especially in high-end leather products. The existing methods have problems of insufficient accuracy and high equipment requirements.
The specific primer KLHL17 forward and reverse primers were designed, combined with PCR amplification and agarose gel electrophoresis technology, for specific identification of sheepskin and amplification of 106 bp of KLHL17 gene.
It realizes fast, simple and accurate sheepskin appraisal, reduces equipment requirements, improves identification efficiency and standardizes the leather market order.
Smart Images
Figure CN120366470A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of leather identification, and in particular relates to a primer, a kit and an identification method for specifically identifying sheepskin. Background Art
[0002] Natural leather, recycled leather, and artificial leather are widely used in our daily life products. Consumers have increasing requirements for product quality, especially the knowledge of leather origin. There have always been clear requirements for the labeling of leather products. However, with the increasing sophistication of processing technology, the difference in appearance between artificial leather and natural leather has gradually narrowed, and it is difficult for consumers to accurately distinguish the characteristics of products. In addition, the quality of leather products on the market is uneven, and it is not uncommon for unscrupulous merchants to replace natural leather with artificial leather in whole or in part, which has led to an increasing demand for leather product identification.
[0003] At present, the research on leather material identification at home and abroad is not systematic. Existing identification methods include microscopy, infrared spectroscopy and DNA identification. Microscopy (such as national standards GB / T38408-2019 and GB / T38416-2019, etc.) can qualitatively identify different types of leather, but its accuracy is limited and its ability to distinguish complex textures is insufficient. Although infrared spectroscopy can analyze the chemical composition of leather, for natural leather, such as common cattle, sheep, pigs, etc., the composition and content of collagen amino acids and elements such as carbon, hydrogen, oxygen, and nitrogen are extremely similar, and no obvious differences can be shown in the infrared spectrum, making it difficult to distinguish. In addition, the anti-interference performance of infrared spectroscopy is relatively poor. The coating materials on the leather surface and the chemical reagents used in the processing process will interfere with the results of infrared spectroscopy. Although the DNA identification method has high accuracy, it relies on qualitative PCR technology and the prejudgment of suspected provenance, and has high requirements for primer specificity, which limits its scope of application.
[0004] Sheep skin occupies an important position in high-end leather products due to its softness and delicate texture, especially vegetable-tanned sheep skin, which is highly respected in the luxury market and handicraft field due to its natural texture and environmental protection characteristics. However, the identification of sheep skin is difficult, and existing technical means cannot meet market demand. Therefore, the development of a fast, simple and scientific identification method, especially for sheep skin, has important application value and is of great significance to regulating the leather market and protecting consumer rights. Summary of the invention
[0005] The purpose of the present invention is to solve the deficiencies in the prior art and provide a primer, a kit and an identification method for specific identification of sheepskin. The method is rapid, simple, accurate, has a wide range of applications and has low requirements on equipment.
[0006] The specific technical solution adopted by the present invention is as follows:
[0007] In the first aspect, the present invention provides a primer for specifically identifying sheepskin, including a KLHL17 forward primer shown in SEQ ID NO.1 and a KLHL17 reverse primer shown in SEQ ID NO.2.
[0008] In the second aspect, the present invention provides a kit for specifically identifying sheepskin, which includes: 2× Fast Taq PCR premix with a volume ratio of 50%, a KLHL17 forward primer shown in SEQ ID NO.1 with a volume ratio of 3% - 5%, a KLHL17 reverse primer shown in SEQ ID NO.2 with a volume ratio of 3% - 5%, and the rest is made up with double-distilled water; wherein the concentrations of the KLHL17 forward primer and the KLHL17 reverse primer are 8 - 12 μM.
[0009] In the third aspect, the present invention provides a method for identifying sheepskin using the primer described in the first aspect, and the specific steps are as follows:
[0010] S1: Extract the DNA of the cortical sample to be detected to obtain a total DNA template;
[0011] S2: Use the KLHL17 forward primer shown in SEQ ID NO.1 and the KLHL17 reverse primer shown in SEQ ID NO.2 to perform a PCR amplification reaction on the total DNA template obtained in step S1 to obtain an amplified DNA fragment;
[0012] S3: Use agarose gel electrophoresis to detect the amplified DNA fragment obtained in step S2 to obtain a PCR amplification band; if the size of the PCR amplification band is 106 bp, then it is determined that the sample is sheepskin.
[0013] Preferably, the system of the PCR amplification reaction described in step S2 is 20 μL, including 1 μL of the total DNA template, 10 μL of 2× Fast Taq PCR premix, 0.6 μL of the KLHL17 forward primer, and 0.6 μL of the KLHL17 reverse primer; wherein the DNA content in the total DNA template is 4% - 40%, and the concentrations of the KLHL17 forward primer and the KLHL17 reverse primer are 8 - 12 μM.
[0014] Further, the system of the PCR amplification reaction described in step S2 is 20 μL, including 1 μL of total DNA template, 10 μL of 2× Fast Taq PCR Premix, 0.6 μL of KLHL17 forward primer, 0.6 μL of KLHL17 reverse primer, and double deionized water is added to 20 μL; wherein the DNA content in the total DNA template is 4% - 40%, and the concentrations of the KLHL17 forward primer and the KLHL17 reverse primer are 10 μM.
[0015] Preferably, the conditions of the PCR amplification reaction in step S2 are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 59°C for 15 s, extension at 72°C for 20 s, for 35 cycles; extension at 72°C for 5 min to obtain the amplified DNA fragment.
[0016] Preferably, the method for detecting by agarose gel electrophoresis in step S3 is as follows: Take 6 μL of the amplified DNA fragment and electrophorese it on a 1.2% agarose gel, and the electrophoresis buffer is 1× TAE, and electrophoresis is carried out at a constant voltage of 120 V for 25 min.
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] The present invention first proposes a primer that can simultaneously amplify the KLHL17 gene of about 106 bp in fresh sheep skin and vegetable-tanned sheep leather, and based on the PCR-gel electrophoresis technology, a method for specifically identifying sheep skin is established. This primer is designed for the KLHL17 gene and can efficiently achieve PCR amplification, ensuring the specificity and stability of the amplification results.
[0019] The method of the present invention has the characteristics of being fast, simple, accurate, wide application range, and low equipment requirements, and can significantly improve the identification efficiency and reduce the detection cost. In addition, the wide application of this method is of great significance for standardizing the order of the leather market and improving the accuracy of leather product traceability. Description of the Drawings
[0020] Figure 1 It is the identification result diagram of 5 kinds of fresh skins in Example 1;
[0021] Figure 2 It is the identification result diagram of vegetable-tanned sheep skin and vegetable-tanned cowhide in Example 2;
[0022] Figure 3 It is the identification result diagram of 5 kinds of fresh skins in Comparative Example 1;
[0023] Figure 4 It is the identification result diagram of vegetable-tanned sheep skin and vegetable-tanned cowhide in Comparative Example 2. Detailed Embodiments
[0024] The present invention will be further described and explained below in conjunction with the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined correspondingly on the premise that there is no conflict with each other.
[0025] Example 1: Screening of KLHL17 protein and design of specific primers
[0026] In this example, primers capable of specifically identifying sheepskin were screened based on the gene sequence of KLHL17 protein, as follows:
[0027] (1) Search for the gene sequences of KLHL17 proteins of sheep, goats, cattle, and pigs in the Uniprot database. Perform protein sequence alignment on the CLUSTALW website to screen out the protein sequences unique to sheep.
[0028] (2) Find the DNA sequence corresponding to the KLHL17 protein of sheep in the NCBI database and download this sequence.
[0029] (3) Import the downloaded DNA sequence into Premier-Primer 5 software for primer design. When designing primers, the following requirements need to be met: the primer length is 18 - 21 bp, the GC content is 40 - 60%, and the bases are distributed as randomly as possible. The primers with the strongest specificity were screened out, including the KLHL17 forward primer shown in SEQ ID NO.1 and the KLHL17 reverse primer shown in SEQ ID NO.2.
[0030] Example 2: Identification of fresh skins
[0031] This example provides a method for identifying fresh skins of 5 kinds of livestock, namely pigs, yellow cattle, buffaloes, goats, and sheep. The specific steps are as follows:
[0032] (1) Collect fresh skins marked as pigs, yellow cattle, buffaloes, goats, and sheep from the market. Use a conventional genomic DNA extraction kit to extract the DNA of each fresh skin sample to obtain the total DNA template.
[0033] (2) Use the KLHL17 forward primer shown in SEQ ID NO.1 and the KLHL17 reverse primer shown in SEQ ID NO.2 to perform PCR amplification reactions on the 5 kinds of total DNA templates obtained in step (1) respectively to obtain amplified DNA fragments.
[0034] The system for the PCR amplification reaction is as follows: 1 μL of total DNA template, 10 μL of 2× Fast Taq PCR Premix, 0.6 μL of KLHL17 forward primer, 0.6 μL of KLHL17 reverse primer, and add ddH2O to 20 μL. The DNA content in the total DNA template is approximately 20%, and the concentrations of the KLHL17 forward primer and the KLHL17 reverse primer are 10 μM. The 2× Fast Taq PCR Premix includes: Taq DNA polymerase, elongation promoting factor, dNTP, green dye, and an optimized buffer system.
[0035] The conditions for the PCR amplification reaction are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 59°C for 15 s, extension at 72°C for 20 s, for 35 cycles; extension at 72°C for 5 min to obtain the amplified DNA fragment.
[0036] (3) The amplified DNA fragments obtained in step (2) were detected by agarose gel electrophoresis respectively, and the method is as follows: Take 6 μL of the amplified DNA fragment and electrophorese it on a 1.2% agarose gel. The electrophoresis buffer is 1× TAE, and electrophoresis is carried out at a constant voltage of 120 V for 25 min. Photograph under a gel imaging system, and the results are as Figure 1 shown. Figure 1 In lanes 1 - 5 are the PCR amplification bands of fresh skin samples of pig, goat, sheep, yellow cattle, and buffalo respectively, and lane M is the nucleic acid molecular weight standard of 2000 bp. It can be clearly seen that the size of the PCR amplification band of fresh sheep skin is approximately 106 bp, and the sizes of the PCR amplification bands of fresh pig, yellow cattle, buffalo, and goat skins are significantly less than 100 bp. This indicates that the identification method provided by the present invention can specifically identify sheep skin.
[0037] Example 3 Identification of vegetable-tanned leather
[0038] This example provides a method for identifying vegetable-tanned sheep leather and vegetable-tanned cattle leather, and the specific steps are as follows:
[0039] (1) Obtain vegetable-tanned sheep leather and vegetable-tanned cattle leather. Use a conventional genomic DNA extraction kit to extract the DNA of the vegetable-tanned leather samples respectively to obtain the total DNA template.
[0040] (2) Use the KLHL17 forward primer shown in SEQ ID NO.1 and the KLHL17 reverse primer shown in SEQ ID NO.2 to perform PCR amplification reactions on the 2 kinds of total DNA templates obtained in step (1) respectively to obtain the amplified DNA fragments.
[0041] The system for the PCR amplification reaction is as follows: 1 μL of total DNA template, 10 μL of 2× Fast Taq PCR Premix, 0.6 μL of KLHL17 forward primer, 0.6 μL of KLHL17 reverse primer, and add ddH2O to 20 μL. The DNA content in the total DNA template is about 20%, and the concentrations of the KLHL17 forward primer and the KLHL17 reverse primer are 10 μM. The 2× Fast Taq PCR Premix includes: Taq DNA polymerase, extension promoting factor, dNTP, green dye, and an optimized buffer system.
[0042] The conditions for the PCR amplification reaction are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 59°C for 15 s, extension at 72°C for 20 s, with 35 cycles; extension at 72°C for 5 min to obtain the amplified DNA fragment.
[0043] (3) The amplified DNA fragments obtained in step (2) were respectively detected by agarose gel electrophoresis as follows: Take 6 μL of the amplified DNA fragment and electrophorese it on a 1.2% agarose gel. The electrophoresis buffer is 1× TAE, and electrophoresis is carried out at a constant voltage of 120 V for 25 min. Photograph under a gel imaging system, and the results are as Figure 2 shown. Figure 2 In lanes 1 and 2 are the PCR amplification bands of vegetable-tanned sheepskin and vegetable-tanned cowhide respectively, and M is the nucleic acid molecular weight standard of 2000 bp. It can be clearly seen that the size of the PCR amplification band of vegetable-tanned sheep leather is about 106 bp, and the size of the PCR amplification band of vegetable-tanned cowhide is significantly less than 100 bp. This shows that even after vegetable-tanning treatment, the identification method provided by the present invention can still specifically identify sheepskin.
[0044] Comparative Example 1 Identification of fresh skins with other primers
[0045] In this comparative example, the XIRP2 forward primer shown in SEQ ID NO.3 and the XIRP2 reverse primer shown in SEQ ID NO.4 were used to identify fresh skins of 5 kinds of livestock including pigs, cattle, buffaloes, goats, and sheep. The specific steps are as follows:
[0046] (1) Fresh skins marked as pigs, cattle, buffaloes, goats, and sheep were collected from the market. The DNA of each fresh skin sample was respectively extracted using a conventional genomic DNA extraction kit to obtain the total DNA template.
[0047] (2) Using the XIRP2 forward primer and the XIRP2 reverse primer, PCR amplification reactions were respectively carried out on the 5 kinds of total DNA templates obtained in step (1) to obtain amplified DNA fragments.
[0048] The system for the PCR amplification reaction is as follows: 1 μL of total DNA template, 10 μL of 2× Fast Taq PCR Premix, 0.6 μL of XIRP2 forward primer, 0.6 μL of XIRP2 reverse primer, and add ddH2O to 20 μL. The DNA content in the total DNA template is about 20%, and the concentrations of the XIRP2 forward primer and the XIRP2 reverse primer are 10 μM. The 2× Fast Taq PCR Premix includes Taq DNA polymerase, extension promoting factor, dNTP, green dye, and an optimized buffer system.
[0049] The conditions for the PCR amplification reaction are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 59°C for 15 s, extension at 72°C for 20 s, for 35 cycles; extension at 72°C for 5 min to obtain the amplified DNA fragment.
[0050] (3) The amplified DNA fragments obtained in step (2) were respectively detected by agarose gel electrophoresis. The method is as follows: Take 6 μL of the amplified DNA fragment and electrophorese it on a 1.2% agarose gel. The electrophoresis buffer is 1× TAE, and electrophoresis is carried out at a constant voltage of 120 V for 25 min. Photograph under a gel imaging system, and the results are as Figure 3 shown. Figure 3 In lanes 1 - 5 are the PCR amplification bands of fresh skin samples of pig, goat, sheep, yellow cattle, and buffalo respectively, and lane M is the nucleic acid molecular weight standard of 2000 bp. It can be seen that there is no obvious difference in the sizes of the PCR amplification bands of fresh skin of sheep, pig, yellow cattle, buffalo, and goat, and they are all less than 100 bp. This shows that it is impossible to specifically identify sheepskin using the XIRP2 forward primer and the XIRP2 reverse primer.
[0051] Comparative Example 2 Identification of vegetable-tanned leather with other primers
[0052] In this comparative example, the XIRP2 forward primer shown in SEQ ID NO.3 and the XIRP2 reverse primer shown in SEQ ID NO.4 were used to identify vegetable-tanned sheep leather and vegetable-tanned cattle leather. The specific steps are as follows:
[0053] (1) Obtain vegetable-tanned sheep leather and vegetable-tanned cattle leather. Use a conventional genomic DNA extraction kit to extract the DNA of the vegetable-tanned leather samples respectively to obtain the total DNA template.
[0054] (2) Use the XIRP2 forward primer and the XIRP2 reverse primer to perform PCR amplification reactions on the 2 kinds of total DNA templates obtained in step (1) respectively to obtain the amplified DNA fragments.
[0055] The system for the PCR amplification reaction is as follows: 1 μL of total DNA template, 10 μL of 2× Fast Taq PCR Premix, 0.6 μL of XIRP2 forward primer, 0.6 μL of XIRP2 reverse primer, and add ddH2O to 20 μL. The DNA content in the total DNA template is about 20%, and the concentrations of the XIRP2 forward primer and the XIRP2 reverse primer are 10 μM. The 2× Fast Taq PCR Premix includes: Taq DNA polymerase, extension promoting factor, dNTP, green dye, and an optimized buffer system.
[0056] The conditions for the PCR amplification reaction are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 59°C for 15 s, extension at 72°C for 20 s, for 35 cycles; extension at 72°C for 5 min to obtain the amplified DNA fragment.
[0057] (3) The amplified DNA fragments obtained in step (2) were respectively detected by agarose gel electrophoresis as follows: Take 6 μL of the amplified DNA fragment and electrophorese it on a 1.2% agarose gel. The electrophoresis buffer is 1× TAE, and electrophoresis is carried out at a constant voltage of 120 V for 25 min. Photograph under a gel imaging system, and the results are as Figure 4 shown. Figure 4 In lanes 1 and 2 are the PCR amplification bands of vegetable-tanned sheepskin and vegetable-tanned cowhide respectively, and M is the nucleic acid molecular weight standard of 2000 bp. It can be seen that there is no obvious difference in the sizes of the PCR amplification bands of vegetable-tanned sheepskin and vegetable-tanned cowhide, and both are less than 100 bp. This shows that the XIRP2 forward primer and the XIRP2 reverse primer cannot specifically identify sheepskin.
[0058] The above-described embodiments are only a preferred solution of the present invention, but they are not intended to limit the present invention. Those of ordinary skill in the relevant technical field can still make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by adopting equivalent replacement or equivalent transformation fall within the protection scope of the present invention.
Claims
1. A primer for specifically identifying sheepskin, characterized in that, It includes the forward primer of KLHL17 shown in SEQ ID NO.1 and the reverse primer of KLHL17 shown in SEQ ID NO.
2.
2. A kit for specifically identifying sheepskin, characterized in that, The kit includes: 2× Fast Taq PCR Premix with a volume ratio of 50%, the forward primer of KLHL17 shown in SEQ ID NO.1 with a volume ratio of 3% - 5%, the reverse primer of KLHL17 shown in SEQ ID NO.2 with a volume ratio of 3% - 5%, and the rest is made up with double-distilled water; wherein the concentrations of the forward primer of KLHL17 and the reverse primer of KLHL17 are 8 - 12 μM.
3. A method for identifying sheepskin using the primer described in claim 1, characterized in that, The specific steps are as follows: S1: Extract the DNA of the cortical sample to be detected to obtain the total DNA template; S2: Use the forward primer of KLHL17 shown in SEQ ID NO.1 and the reverse primer of KLHL17 shown in SEQ ID NO.2 to perform a PCR amplification reaction on the total DNA template obtained in step S1 to obtain an amplified DNA fragment; S3: Use agarose gel electrophoresis to detect the amplified DNA fragment obtained in step S2 to obtain a PCR amplification band; if the size of the PCR amplification band is 106 bp, then judge that the sample is sheepskin.
4. The method for identifying sheepskin according to claim 3, characterized in that, The system of the PCR amplification reaction described in step S2 is 20 μL, including 1 μL of total DNA template, 10 μL of 2× Fast Taq PCR Premix, 0.6 μL of the forward primer of KLHL17, and 0.6 μL of the reverse primer of KLHL17; wherein the DNA content in the total DNA template is 4% - 40%, and the concentrations of the forward primer of KLHL17 and the reverse primer of KLHL17 are 8 - 12 μM.
5. The method for identifying sheepskin according to claim 4, characterized in that, The system of the PCR amplification reaction described in step S2 is 20 μL, including 1 μL of total DNA template, 10 μL of 2× Fast Taq PCR Premix, 0.6 μL of the forward primer of KLHL17, 0.6 μL of the reverse primer of KLHL17, and double-deionized water is added to 20 μL; wherein the DNA content in the total DNA template is 4% - 40%, and the concentrations of the forward primer of KLHL17 and the reverse primer of KLHL17 are 10 μM.
6. The method for identifying sheepskin according to claim 3, characterized in that, The conditions of the PCR amplification reaction described in step S2 are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 59°C for 15 s, extension at 72°C for 20 s, for 35 cycles; extension at 72°C for 5 min to obtain an amplified DNA fragment.
7. The method for identifying sheepskin according to claim 3, wherein The method for agarose gel electrophoresis detection described in step S3 is as follows: Take 6 μL of the amplified DNA fragment and electrophorese it on a 1.2% agarose gel, the electrophoresis buffer is 1× TAE, and electrophoresis is carried out at a constant voltage of 120 V for 25 min.