A primer, a kit and an identification method for identifying a leather cultural relic species
By designing universal primers based on cytochrome b (cytb) gene fragments and combining them with PCR amplification and sequencing methods, the problem of species identification of leather artifacts has been solved. This enables rapid, simple, and accurate identification of aged and environmentally damaged leather artifacts, and is applicable to the identification of leather artifacts in archaeological work.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2024-07-01
- Publication Date
- 2026-04-28
AI Technical Summary
Existing technologies are insufficient to efficiently and accurately identify the species origin of leather artifacts damaged by aging and environmental factors, especially cow and sheep leather artifacts, and commonly used methods may cause secondary damage to the artifacts.
A universal primer based on the cytochrome b (cytb) gene fragment of animal mitochondria was designed. The primers cytbF and cytbR were used to identify the DNA of leather artifacts by PCR amplification and sequencing, and the species was determined by BLAST similarity comparison.
It can quickly, easily, and accurately identify single or mixed aged and environmentally damaged cattle and sheep leather artifacts, enabling precise identification of sheep, goats, cattle, and buffalo, and is suitable for identifying leather artifacts that are difficult to identify in archaeological work.
Smart Images
Figure CN118667935B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular archaeology of leather artifacts, specifically relating to primers, reagent kits, and identification methods for identifying species and genera of leather artifacts. Background Technology
[0002] Leather, as an important raw material for production and daily life in ancient societies, is a crucial component of cultural relics. Identifying and analyzing unearthed leather artifacts yields invaluable archaeological information, providing crucial reference for subsequent conservation efforts. Traditionally, leather was primarily sourced from animals such as cattle and sheep, processed using vegetable tanning techniques to produce leather products. Leather artifacts, whether buried underground for extended periods or continuously exposed to sunlight, are susceptible to degradation due to environmental factors such as light, heat, humidity, and microorganisms. The surface morphology is rarely preserved intact, and the internal structure may be damaged. In more severe cases, only traces of leather may remain in the soil, barely visible to the naked eye, before eventually decaying and disappearing.
[0003] The severe deterioration of leather artifacts poses significant challenges to identifying their species origin. While some commonly used modern analytical equipment and methods, such as surface analysis and infrared spectroscopy, can provide preliminary identification of the species of leather artifacts, the rarity and preciousness of these artifacts, along with the severity of their deterioration, necessitate avoiding secondary damage during testing. Furthermore, the complex environment in which leather artifacts exist makes them prone to mixing with other environmental components, all of which can reduce the accuracy of identification and prevent precise determination of the leather's origin. Therefore, to address these challenges, new methods are needed to identify the origin of leather artifacts.
[0004] With the development of molecular biology, we can rely on highly species-specific DNA information to identify species through a series of processes, including DNA extraction, PCR amplification, and sequencing. The tanning process and environmental acid-base conditions can damage leather DNA, making it difficult to extract complete DNA from tanned, deteriorated leather, thus hindering identification. However, cytochrome b (cytb) gene fragments in mitochondrial DNA (mtDNA) are more likely to be preserved than nuclear DNA and can be detected by polymerase chain reaction (PCR). Currently, methods for simultaneously identifying leather from multiple species using universal primers have not been reported, indicating that this field requires further research and exploration. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and to provide primers, reagent kits and identification methods for identifying species of leather writing.
[0006] The specific technical solution adopted in this invention is as follows:
[0007] In a first aspect, the present invention provides primers for identifying species and genera of leather writing, including upstream primer cytbF as shown in SEQ ID NO.1 and downstream primer cytbR as shown in SEQ ID NO.2.
[0008] Secondly, the present invention provides a method for identifying the species and genera of leather using the primers described in the first aspect for identifying leather species and genera, the specific steps of which are as follows:
[0009] S1: Extract DNA from the leather sample to be tested to obtain the total DNA template;
[0010] S2: Using the upstream primer cytbF shown in SEQ ID NO.1 and the downstream primer cytbR shown in SEQ ID NO.2, the total DNA template obtained in step S2 was subjected to PCR amplification to obtain the amplification product;
[0011] S3: Perform bidirectional sequencing or next-generation sequencing on the amplification products obtained in step S2. Compare the sequence information of the leather to be tested with the BLAST similarity sequences of sheep cytb gene, goat cytb gene, cattle cytb gene, and buffalo cytb gene to determine the species of the leather sample to be tested.
[0012] Preferably, the PCR amplification reaction system in step S2 includes: upstream primer cytbF 0.4 μmol / L, downstream primer cytbR 0.4 μmol / L, total DNA template 20–100 ng / uL, Taq DNA polymerase 0.2 U / uL, deoxyribonucleoside triphosphate (dNTP) 0.4 mmol / L, MgCl2 1 mmol / L, and 2×Taq buffer.
[0013] Furthermore, the PCR amplification reaction system described in step S2 is as follows: 12.5 μL of 2×Taq PCR Mix, 1 μL of 10 μmol / L upstream primer cytbF, 1 μL of 10 μmol / L downstream primer cytbR, 2.0 μL of total DNA template, and water added to 25 μL; wherein the 2×Taq PCR Mix contains: 0.2 U / μL of Taq DNA polymerase, 0.4 mmol / L of deoxyribonucleoside triphosphate dNTP, 1 mmol / L of MgCl2, and 2×Taq buffer.
[0014] Preferably, the PCR amplification reaction conditions in step S2 are: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 5 s, for 30 cycles; 72℃ final extension for 5 min.
[0015] Preferably, if the leather sample to be tested is a mixed leather sample, then the amplification product is subjected to next-generation sequencing in step S3; a mixed leather sample refers to a situation where multiple leathers are mixed together and cannot be separated or the number of species in the obtained sample cannot be determined.
[0016] Preferably, if the leather sample to be tested is a single leather sample, then the amplification product is subjected to bidirectional sequencing in step S3.
[0017] Preferably, in step S3, if the sequence information of the leather to be tested obtained by bidirectional sequencing has a homology of >96% with the barcode sequence of the sheep cytb gene, then the leather sample is determined to be sheep leather; if the sequence information of the leather to be tested obtained by bidirectional sequencing has a homology of >96% with the barcode sequence of the goat cytb gene, then the leather sample is determined to be goat leather; if the sequence information of the leather to be tested obtained by bidirectional sequencing has a homology of >96% with the barcode sequence of the cattle cytb gene, then the leather sample is determined to be cattle leather; if the sequence information of the leather to be tested obtained by bidirectional sequencing has a homology of >96% with the barcode sequence of the buffalo cytb gene, then the leather sample is determined to be buffalo leather.
[0018] Thirdly, the present invention provides a kit for identifying the species of leather specimens, wherein the components of the kit constitute a 25 μL PCR amplification reaction system, comprising the upstream primer cytbF 0.4 μmol / L, the downstream primer cytbR 0.4 μmol / L, Taq DNA polymerase 0.2 U / μL, deoxyribonucleoside triphosphate dNTP 0.4 mmol / L, MgCl2 1 mmol / L, and 2×Taq buffer as described in claim 1.
[0019] Fourthly, the present invention provides an application of the identification method described in the second aspect in the identification of species and genera of leather writing.
[0020] Compared with the prior art, the present invention has the following advantages:
[0021] This invention presents for the first time a universal primer designed based on a fragment of the animal mitochondrial cytochrome b (cytb) gene. Using this universal primer, a 119 bp DNA barcode sequence can be amplified, enabling the successful identification of sheep, goat, cattle, and buffalo leather artifacts that are difficult to identify due to aging and disfigurement. This primer is applicable not only to leather samples from a single species but also to mixed leather samples.
[0022] The primers provided by this invention can also identify leather artifacts made from sheep, goats, cattle, and buffalo that have been damaged by acids and alkalis in the environment. It can not only successfully identify leather artifacts that are difficult to identify due to aging and external damage, but also precisely subdivide the two major leather-producing species, cattle and sheep, down to the species level: cattle and buffalo, and sheep and goats.
[0023] The identification method provided by this invention can quickly, easily, and accurately identify the species and origin of leather artifacts, providing a new identification method for samples of mixed leather artifacts that only leave traces in archaeological work. It has great significance in the fields of artifact identification, protection, and restoration. Attached Figure Description
[0024] Figure 1 The results of gel electrophoresis of total DNA from sheep, goat, cattle, and buffalo leather samples in Example 1 are shown. The Maker fragment sizes are 600, 500, 400, 300, 200, and 100, respectively. Lane 1 contains total DNA from sheep leather artifacts, lane 2 from goat leather artifacts, lane 3 from cattle leather artifacts, and lane 4 from buffalo leather artifacts.
[0025] Figure 2 The images show the PCR amplification electrophoresis results of DNA samples from sheep, goat, cattle, and buffalo leather samples in Example 1. The Marker fragment sizes are 600, 500, 400, 300, 200, and 100, respectively. Lane 1 shows the PCR product from sheep leather artifacts, lane 2 from goat leather artifacts, lane 3 from cattle leather artifacts, lane 4 from buffalo leather artifacts, and lane 5 shows the negative control result obtained using deionized water as the amplification template.
[0026] Figure 3 The image shows the bidirectional sequencing results of sheep, goat, cattle, and buffalo leather samples from Example 1, where A represents sheep leather sample, B represents goat leather sample, C represents cattle leather sample, and D represents buffalo leather sample.
[0027] Figure 4 This is a pie chart showing the relative abundance of dominant species in the second-generation sequencing of the leather mixture sample in Example 2.
[0028] Figure 5 The image shows the bidirectional sequencing results of acid-aged sheep, goat, cattle, and buffalo leather samples from Example 3. A represents acid-aged sheep leather, B represents acid-aged goat leather, C represents acid-aged cattle leather, and D represents acid-aged buffalo leather.
[0029] Figure 6The images show the bidirectional sequencing results of alkali-aged sheep, goat, cattle, and buffalo leather samples from Example 4. A represents the alkali-aged sheep leather sample, B represents the alkali-aged goat leather sample, C represents the alkali-aged cattle leather sample, and D represents the alkali-aged buffalo leather sample.
[0030] Figure 7 This is a peak diagram of the bidirectional sequencing results of the leather sample to be tested in Example 5. Detailed Implementation
[0031] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.
[0032] Example 1
[0033] 1. Genetic marker selection and primer design
[0034] The cytochrome b (cytb) gene from the mitochondrial DNA of sheep (Ovis aries) NC_001941.1, goat (Caprahircus) NC_005044.2, cattle (Bos tauruscattle) NC_006853.1, and buffalo (Bubalus bubalis) MK499431.1, published in the Genbank database, was downloaded and subjected to multiple sequence alignment. Primers were designed using the biological software Oligo.7 targeting the conserved regions common to the four species. Primers without hairpin structures or dimers, with a Tm value of around 60℃ and a high score were selected.
[0035] Degenerate primers were designed to target differences of a few bases between sequences from different species, but the number of degenerate codons should not exceed three. Multiple pairs of universal primers were designed. The universal primers were then preliminarily screened and validated for their universality using NCBI's Primer-BLAST algorithm. These universal primer pairs must meet the following requirements: the PCR amplification bands must be free of impurities and clear, and they must be able to identify four species.
[0036] 2. Total DNA template extraction
[0037] 100 mg of leather samples from sheep, goats, cattle, and buffalo were taken respectively, and DNA was extracted using a standard genomic DNA extraction kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The specific procedure was as follows: 200 μL of lysis buffer was added, followed by 20 μL of 20 mg / mL proteinase K. After mixing, the mixture was incubated at 56 °C for 24 h. 200 μL of extraction buffer was added, and the mixture was incubated at 50 °C for 40 min. 200 μL of anhydrous ethanol was added, and the mixture was thoroughly shaken for 30 s. The resulting solution and flocculent precipitate were added to a silica gel membrane adsorption column, centrifuged (12000 rpm, 30 s), and the waste liquid was discarded. 500 μL of elution buffer was added to the adsorption column, centrifuged (12000 rpm, 30 s), and the waste liquid was discarded. Add 500 μL of elution buffer to the adsorption column, centrifuge (12000 rpm, 30 s) and discard the waste liquid, then centrifuge again (12000 rpm, 2 min) and discard the waste liquid. Incubate the adsorption column at room temperature for 3 min, add 50 μL of elution buffer to the middle of the adsorption membrane, incubate at room temperature for 3 min, then centrifuge (12000 rpm, 2 min). The resulting solution is the total DNA template.
[0038] A 1 μL sample of genomic DNA template was tested on an ultra-micro spectrophotometer. The quality of the extracted genomic DNA template was evaluated by comparing the absorbance and peak patterns at 260 nm and 280 nm, the A260 / A280 ratio, and the concentration of DNA in the sample, combined with the agarose gel electrophoresis results of the genomic DNA sample. Figure 1 The results of total DNA gel electrophoresis from sheep, goat, cattle, and buffalo leather samples.
[0039] 3. PCR amplification
[0040] Using the extracted total DNA as a template, the specificity of several designed universal primers was verified. The designed universal primers were used to perform PCR amplification on the total DNA templates from sheep leather, goat leather, cattle leather, and buffalo leather samples, respectively, yielding PCR amplification products for sheep leather, goat leather, cattle leather, and buffalo leather.
[0041] The PCR amplification reaction system was 25 μL: 12.5 μL of 2×Taq PCR Mix, 1 μL of 10 μmol / L upstream primer cytbF, 1 μL of 10 μmol / L downstream primer cytbR, 2.0 μL of total DNA template, and ddH2O added to 25 μL; the 2×Taq PCR Mix contained: 0.2 U / μL Taq DNA polymerase, 0.4 mmol / L deoxyribonucleoside triphosphate (dNTP), 1 mmol / L MgCl2, and 2×Taq buffer.
[0042] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 5 s, for 30 cycles; and 72℃ extension for 5 min.
[0043] 4. Detection of PCR amplification products by agarose electrophoresis
[0044] After the PCR amplification reaction was completed, the size of the PCR amplification products was detected by agarose electrophoresis: 5 μL of PCR amplification products of sheep leather, goat leather, cattle leather and buffalo leather were electrophoresed on a 2% agarose gel with 1×TAE electrophoresis buffer and electrophoresis at 120V constant voltage for 20 min. The gel was then photographed using a gel imaging system.
[0045] Primers were selected that amplified total DNA templates from sheep, cattle, buffalo, and goat leather samples without any impurities, and that successfully amplified clear bands from all four DNA types. The results are as follows: Figure 2 As shown. The primer sequence was determined to be:
[0046] Upstream primer cytbF: 5'-GGAGACCCWGACAACTAYACC-'3'.
[0047] Downstream primer cytbR: 5'-CCTCCTAGTTTGTTRGGRA-'3'.
[0048] Where W is A or T, Y is C or T, and R is A or G.
[0049] like Figure 2 As shown, the band sizes of the PCR amplification products of sheep leather, goat leather, cattle leather, and buffalo leather obtained by PCR amplification using upstream primer cytbF and downstream primer cytbR are all approximately 119 bp.
[0050] The nucleotide sequences of the PCR amplification products of sheep leather are shown in SEQ ID NO.3, those of goat leather are shown in SEQ ID NO.4, those of cattle leather are shown in SEQ ID NO.5, and those of buffalo leather are shown in SEQ ID NO.6.
[0051] 5. Sequencing and Identification
[0052] The PCR amplification products of sheep leather, goat leather, cattle leather, and buffalo leather were sent to a sequencing company (Shanghai Sangon Biotech) for bidirectional sequencing, and the sequencing results were obtained as follows: Figure 3 As shown. The obtained sequencing results were spliced and the primer sequences at both ends were deleted to obtain the sequence information for sheep leather, goat leather, cattle leather, and buffalo leather, respectively. The sequencing results were then used for species identification using the BLAST-Based Method. Figure 3 As can be seen, the sequencing peaks are clear and non-overlapping, and the displayed bases can be used for sequence alignment.
[0053] The spliced base sequences were input into NCBI for online BLAST homology comparison, and the results are shown in Table 1. The BLAST similarity analysis of sheep leather sequences with the sheep cytb gene barcode sequence showed 96.25% homology; the sequence of goat leather sequences with the goat cytb gene barcode sequence showed 96.92% homology; the sequence of cattle leather sequences with the cattle cytb gene barcode sequence showed 100.00% homology; and the sequence of buffalo leather sequences with the buffalo cytb gene barcode sequence showed 97.47% homology. The homology of all the above samples was greater than 96%. This indicates that the products obtained by PCR amplification using upstream primer cytbF and downstream primer cytbR can specifically identify four species: sheep, goat, cattle, and buffalo.
[0054] Table 1. Homology comparison results of samples in Example 1
[0055] Sample Name BLAST result Latin name homology sheep Ovis aries 96.25％ goat Capra hircus 96.92％ cattle Bos taurus 100.00％ buffalo Bubalusbubalis 97.47％
[0056] Example 2
[0057] This embodiment provides a method for identifying mixed leather samples using primers obtained in Example 1, as detailed below:
[0058] 1. Mix four types of leather: sheep leather, goat leather, cow leather, and buffalo leather to obtain a mixed sample.
[0059] 2. Extract DNA from the mixed sample to obtain the total DNA template. The specific extraction method is as described in step 2 of Example 1.
[0060] 3. Using the upstream primer cytbF shown in SEQ ID NO.1 and the downstream primer cytbR shown in SEQ ID NO.2, perform PCR amplification on the total DNA template obtained in step 2 to obtain the amplification product.
[0061] The PCR amplification reaction system was 25 μL: 12.5 μL of 2×Taq PCR Mix, 1 μL of 10 μmol / L upstream primer cytbF, 1 μL of 10 μmol / L downstream primer cytbR, 2.0 μL of total DNA template, and ddH2O added to 25 μL; the 2×Taq PCR Mix contained: 0.2 U / μL Taq DNA polymerase, 0.4 mmol / L deoxyribonucleoside triphosphate (dNTP), 1 mmol / L MgCl2, and 2×Taq buffer.
[0062] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 5 s, for 30 cycles; and 72℃ extension for 5 min.
[0063] 4. Send the amplified product obtained in step 3 to a sequencing company (Shanghai Sangon Biotech) for next-generation sequencing. Analyze the results of the next-generation sequencing analysis report. The results are as follows: Figure 4 As shown.
[0064] The pie chart showing the relative abundance of dominant species indicates the presence of four species in the mixed sample: sheep (Ovis aries), goats (Capra hircus), cattle (Bos tauruscattle), and buffalo (Bubalus bubalis), consistent with the results for sheep, goats, cattle, and buffalo in the DNA mixed sample. This demonstrates that the upstream primer cytbF and downstream primer cytbR provided in this invention can also be used for the detection of mixed leather samples.
[0065] Example 3
[0066] This embodiment provides a method for identifying acid-aged leather samples using primers obtained in Example 1, as detailed below:
[0067] 1. Acid aging of leather samples
[0068] Sheep, goat, cattle, and buffalo leather samples were immersed in 0.5M acetic acid and treated at 4°C for 3 hours. The samples were then neutralized with 0.7M NaCl until neutral. The neutralized sheep, goat, cattle, and buffalo leather samples were rinsed with running water and then stored in pure water for subsequent DNA extraction.
[0069] 2. Extract DNA from sheep, goat, cattle, and buffalo leather samples respectively to obtain total DNA templates. The specific extraction method is as described in step 2 of Example 1.
[0070] 3. Using the upstream primer cytbF shown in SEQ ID NO.1 and the downstream primer cytbR shown in SEQ ID NO.2, perform PCR amplification on the total DNA template obtained in step 2 to obtain the amplification product.
[0071] The PCR amplification reaction system was 25 μL: 12.5 μL of 2×Taq PCR Mix, 1 μL of 10 μmol / L upstream primer cytbF, 1 μL of 10 μmol / L downstream primer cytbR, 2.0 μL of total DNA template, and ddH2O added to 25 μL; the 2×Taq PCR Mix contained: 0.2 U / μL Taq DNA polymerase, 0.4 mmol / L deoxyribonucleoside triphosphate (dNTP), 1 mmol / L MgCl2, and 2×Taq buffer.
[0072] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 5 s, for 30 cycles; and 72℃ extension for 5 min.
[0073] 4. The amplification products obtained in step 3 were sent to a sequencing company (Shanghai Sangon Biotech) for bidirectional sequencing. The bidirectional sequencing results are as follows: Figure 5 The sequencing peaks are clear and non-overlapping, and the displayed bases can be used for sequence alignment.
[0074] The bidirectional sequencing results were subjected to BLAST similarity analysis, and the results are shown in Table 2. The BLAST similarity analysis of the sequence information of acid-aged sheep leather samples with the sheep cytb gene barcode sequence showed a homology of 97.22%; the BLAST similarity analysis of the sequence information of acid-aged goat leather with the goat cytb gene barcode sequence showed a homology of 96.92%; the BLAST similarity analysis of the sequence information of acid-aged cattle leather with the cattle cytb gene barcode sequence showed a homology of 100.00%; and the BLAST similarity analysis of the sequence information of acid-aged buffalo leather with the buffalo cytb gene barcode sequence showed a homology of 98.59%. The homology of all the above samples was greater than 96%. This indicates that the products obtained by PCR amplification using upstream primer cytbF and downstream primer cytbR can specifically identify acid-aged sheep, goat, cattle, and buffalo leather samples.
[0075] Table 2. Homology Comparison Results of Samples in Example 3 (Example 3)
[0076] Sample Name BLAST result Latin name homology Sheep acid aging Ovis aries 97.22％ Goat acid aging Capra hircus 96.92％ Taurine aging Bos taurus 100.00％ Buffalo acid aging Bubalus bubalis 98.59％
[0077] Example 4
[0078] This embodiment provides a method for identifying alkali-aged leather samples using primers obtained in Example 1, as detailed below:
[0079] 1. Alkali aging of leather samples
[0080] Sheep, goat, cattle, and buffalo leather samples were immersed in solutions containing 4% Ca(OH)₂ and 0.5% NaOH, respectively, and treated at 25°C for 3 hours. The samples were then neutralized with 1% (NH₄)₂SO₄ until neutral. The neutralized sheep, goat, cattle, and buffalo leather samples were rinsed with running water and then stored in pure water for subsequent DNA extraction.
[0081] 2. Extract DNA from sheep, goat, cattle, and buffalo leather samples respectively to obtain total DNA templates. The specific extraction method is as described in step 2 of Example 1.
[0082] 3. Using the upstream primer cytbF shown in SEQ ID NO.1 and the downstream primer cytbR shown in SEQ ID NO.2, perform PCR amplification on the total DNA template obtained in step 2 to obtain the amplification product.
[0083] The PCR amplification reaction system was 25 μL: 12.5 μL of 2×Taq PCR Mix, 1 μL of 10 μmol / L upstream primer cytbF, 1 μL of 10 μmol / L downstream primer cytbR, 2.0 μL of total DNA template, and ddH2O added to 25 μL; the 2×Taq PCR Mix contained: 0.2 U / μL Taq DNA polymerase, 0.4 mmol / L deoxyribonucleoside triphosphate (dNTP), 1 mmol / L MgCl2, and 2×Taq buffer.
[0084] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 5 s, for 30 cycles; and 72℃ extension for 5 min.
[0085] 4. The amplification products obtained in step 3 were sent to a sequencing company (Shanghai Sangon Biotech) for bidirectional sequencing. The bidirectional sequencing results are as follows: Figure 6 The sequencing peaks are clear and non-overlapping, and the displayed bases can be used for sequence alignment.
[0086] The bidirectional sequencing results were subjected to BLAST similarity analysis, and the results are shown in Table 3. The sequence information of alkali-aged sheep leather samples showed 98.55% homology with the sheep cytb gene barcode sequence; the sequence information of acid-aged goat leather showed 96.43% homology; the sequence information of acid-aged cattle leather showed 100.00% homology; and the sequence information of acid-aged buffalo leather showed 100.00% homology. All the samples showed homology greater than 96%. This indicates that the products obtained by PCR amplification using upstream primer cytbF and downstream primer cytbR can specifically identify alkali-aged sheep, goat, cattle, and buffalo leather samples.
[0087] Table 3. Homology Comparison Results of Samples in Example 3
[0088] Sample Name BLAST result Latin name homology sheep alkali aging Ovis aries 98.55％ Goat alkali aging Capra hircus 96.43％ Aging of yellow bovine alkali Bos taurus 100.00％ buffalo alkali aging Bubalus bubalis 100.00％
[0089] Example 5
[0090] This embodiment provides a method for identifying leather samples of unknown species using primers obtained in Example 1, as detailed below:
[0091] 1. Take 5mg of leather sample of unknown species, rinse the surface dust and impurities with deionized water, and store it in pure water for subsequent DNA extraction.
[0092] 2. Extract DNA from sheep, goat, cattle, and buffalo leather samples respectively to obtain total DNA templates. The specific extraction method is as described in step 2 of Example 1.
[0093] 3. Using the upstream primer cytbF shown in SEQ ID NO.1 and the downstream primer cytbR shown in SEQ ID NO.2, perform PCR amplification on the total DNA template obtained in step 2 to obtain the amplification product.
[0094] The PCR amplification reaction system was 25 μL: 12.5 μL of 2×Taq PCR Mix, 1 μL of 10 μmol / L upstream primer cytbF, 1 μL of 10 μmol / L downstream primer cytbR, 2.0 μL of total DNA template, and ddH2O added to 25 μL; the 2×Taq PCR Mix contained: 0.2 U / μL Taq DNA polymerase, 0.4 mmol / L deoxyribonucleoside triphosphate (dNTP), 1 mmol / L MgCl2, and 2×Taq buffer.
[0095] The PCR amplification conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 5 s, for 30 cycles; and 72℃ extension for 5 min.
[0096] 4. The amplification products obtained in step 3 were sent to a sequencing company (Shanghai Sangon Biotech) for bidirectional sequencing. The result peak diagram is shown below. Figure 7 As shown in the figure. The bidirectional sequencing results were spliced and organized to obtain the nucleotide sequence of the unknown leather sample as shown in SEQ ID NO.7. The sequencing results were subjected to BLAST homology comparison, and the result showed that the homology with Ovis aries (sheep) was 96.67%, and the leather sample was determined to be sheep leather.
[0097] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.
Claims
1. A method for identifying the species and genus of leathergrass, characterized in that, The specific steps are as follows: S1: Extract DNA from the leather sample to be tested to obtain the total DNA template; S2: Using the upstream primer cytbF shown in SEQ ID NO.1 and the downstream primer cytbR shown in SEQ ID NO.2, the total DNA template obtained in step S1 was subjected to PCR amplification to obtain the amplification product; S3: Perform bidirectional sequencing or next-generation sequencing on the amplification products obtained in step S2. Compare the sequence information of the leather to be tested with the BLAST similarity sequences of sheep cytb gene, goat cytb gene, cattle cytb gene, and buffalo cytb gene to determine the species of the leather sample to be tested.
2. The method for identifying the species and genus of leather texts according to claim 1, characterized in that, The PCR amplification reaction system described in step S2 includes: upstream primer cytbF 0.4 µmol / L, downstream primer cytbR 0.4 µmol / L, total DNA template 20~100 ng / uL, Taq DNA polymerase 0.2 U / uL, deoxyribonucleoside triphosphate dNTP 0.4 mmol / L, MgCl2 1 mmol / L, and 2×Taq buffer.
3. The method for identifying the species and genus of leather texts according to claim 2, characterized in that, The PCR amplification reaction system described in step S2 is as follows: 12.5 µL of 2×Taq PCR Mix, 1 µL of 10 µmol / L upstream primer cytbF, 1 µL of 10 µmol / L downstream primer cytbR, 2.0 µL of total DNA template, and water added to 25 µL. The 2×Taq PCR Mix contains: Taq DNA polymerase 0.2 U / uL, deoxyribonucleoside triphosphate (dNTP) 0.4 mmol / L, MgCl2 1 mmol / L, and 2×Taq buffer.
4. The method for identifying the species and genus of leather texts according to claim 1, characterized in that, The PCR amplification reaction conditions described in step S2 are as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 5 s, for 30 cycles; 72℃ final extension for 5 min.
5. The method for identifying the species and genus of leather texts according to claim 1, characterized in that, If the leather sample to be tested is a mixed leather sample, then in step S3, the amplification product is subjected to next-generation sequencing.
6. The method for identifying the species and genus of leather texts according to claim 1, characterized in that, If the leather sample to be tested is a single leather sample, then the amplification product is subjected to bidirectional sequencing in step S3.
7. The method for identifying the species and genus of leather texts according to claim 1, characterized in that, In step S3, if the sequence information of the leather to be tested obtained by bidirectional sequencing has a homology of >96% with the sheep cytb gene barcode sequence, then the leather sample is determined to be sheep leather; if the sequence information of the leather to be tested obtained by bidirectional sequencing has a homology of >96% with the goat cytb gene barcode sequence, then the leather sample is determined to be goat leather; if the sequence information of the leather to be tested obtained by bidirectional sequencing has a homology of >96% with the cattle cytb gene barcode sequence, then the leather sample is determined to be cattle leather; if the sequence information of the leather to be tested obtained by bidirectional sequencing has a homology of >96% with the buffalo cytb gene barcode sequence, then the leather sample is determined to be buffalo leather.
8. A kit for identifying the species and genus of leather specimens, characterized in that, The components of the kit constitute a 25 µL PCR amplification reaction system: It includes the upstream primer cytbF 0.4 µmol / L as shown in SEQ ID NO.1, the downstream primer cytbR 0.4 µmol / L as shown in SEQ ID NO.2, Taq DNA polymerase 0.2 U / µL, deoxyribonucleoside triphosphate dNTP 0.4 mmol / L, MgCl2 1 mmol / L, and 2×Taq buffer.
9. An application of the identification method according to any one of claims 1 to 7 in the identification of species and genera of leather writing.