Primer probe group, kit and identification method for identifying pterocarpus santalinus
By using TXZT specific primers and fluorescent PCR technology in the identification of sandalwood rosewood, the problems of long time, high misjudgment rate and high destructiveness of sandalwood rosewood in the existing technology are solved, and fast, accurate and small destructive are achieved. detection effect.
Patent Information
- Application Number
- CN202411660071.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-01-07
AI Technical Summary
The prior art is difficult to quickly, accurately and without destructively identify sandalwood and dyed sandalwood, which leads to market confusion and illegal trade, and the traditional method has a high misjudgment rate, long time and is highly destructive to wood.
Provide a primer probe set including TXZT specific primer pairs and TXZT probes. Combined with fluorescent PCR technology, the specific nucleotide sequence design can achieve rapid, accurate and high sensitivity detection of Sandalwood DNA, suitable for use The fluorescence reporter and quenching group labeling were designed to design specific primers and probes for rapid identification of santorinium santorin.
The rapid, accurate and small damage detection of sandalwood and red sandalwood is achieved, which reduces the misjudgment rate, significantly improves the detection efficiency, and reduces the destructiveness of the wood, ensuring the reliability of the identification results.
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Figure CN119265346A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of molecular biological detection for identification of wood species, and in particular to a primer probe set, a kit and an identification method for identifying Pterocarpus santalinus. Background Art
[0002] Pterocarpus santalinus L.f., also known as red sandalwood or small-leaf red sandalwood, is a superior raw material for making furniture and handicrafts, and has always been used as a precious material for producing furniture or handicrafts.
[0003] The demand for red sandalwood products is also growing; however, red sandalwood trees grow very slowly, usually taking more than a hundred years to mature, and due to continuous logging, natural red sandalwood forests are very rare. The sharp increase in people's demand for red sandalwood and its wood supply are in conflict, resulting in a shortage of red sandalwood wood in the market in recent years. Therefore, cheap dye red sandalwood (P.tinctorius) wood imported from Africa has appeared on the market. The market calls it "blood sandalwood" or "African small-leaf red sandalwood". The macroscopic and anatomical characteristics of its wood are very similar to those of Indian red sandalwood, and the English product names red sanders and red sandalwood are consistent with red sandalwood, making it difficult to distinguish. Some unscrupulous vendors sell dye red sandalwood as red sandalwood products; some technical inspection agencies even misjudge dye red sandalwood as red sandalwood, causing huge economic losses to users, seriously infringing on the legitimate rights and interests of consumers and disrupting the normal market competition order. From the perspective of protecting consumer interests and maintaining normal market order, accurately identifying red sandalwood and dye red sandalwood has become a top priority.
[0004] In addition, red sandalwood trees grow very slowly. Due to years of logging, the number of natural red sandalwood forests has become very scarce. Therefore, the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) has included it in Appendix II to regulate its international trade and impose strict restrictions on the international trade of red sandalwood. Some timber importers falsely report red sandalwood as dye red sandalwood when entering the customs, thereby evading high tariffs and restrictions of the CITES Convention. Therefore, from the perspective of maintaining normal market order, protecting wild red sandalwood forests, and combating illegal logging and illegal timber trade, it is of great significance to correctly distinguish between red sandalwood and dye red sandalwood.
[0005] GB / T18107-2000 stipulates that red sandalwood belongs to rosewood, and its identification method is based on simple and practical macroscopic characteristics (such as density, structure, wood color and texture, etc.), supplemented by necessary wood anatomical characteristics to determine its genus. The specific detection process is as follows: 1) Macroscopic inspection mainly tests its wood color, smell, texture, density and other indicators; 2) Wood anatomical characteristics inspection mainly saws the sample into a 1 cubic centimeter cube with three cross-sections on the outer surface: transverse, radial and chord, and then softens the small rosewood block for about 1 to 7 days, then fixes the softened small wood block on the slicer for slicing, then dehydrates and seals the slices, and finally observes the wood anatomical characteristics under an optical microscope.
[0006] The main disadvantages of using traditional methods to inspect the above-mentioned woods are: 1) The inspection time is long. Traditional methods require a combination of macroscopic inspection and wood anatomical characteristics. The macroscopic inspection of wood 12% air-dry density test takes about 3-7 days; the softening, slicing, dehydration, sealing and other processes in the wood anatomical characteristics inspection take 2-7 days. 2) The inspection is destructive and requires a lot of materials. The traditional method density test requires at least 3 pieces of 2cm×2cm×2cm wood blocks; the wood anatomical characteristics inspection requires a piece of wood of 3cm×3cm×1cm in size, which causes serious damage to mahogany products. 3) For wood with similar macroscopic and anatomical structures, the misjudgment rate is high. Summary of the invention
[0007] In view of the shortcomings of the prior art, the purpose of the embodiments of the present application includes providing a primer probe set, a kit and an identification method for identifying Pterocarpus santalinus, which can perform rapid, accurate and less destructive detection and identification of Pterocarpus santalinus with specificity and high sensitivity.
[0008] In the first aspect, the embodiments of the present application provide a primer-probe set for identifying Pterocarpus santalinus, comprising a TXZT-specific primer pair and a TXZT probe, the TXZT-specific primer pair comprising a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence as shown in SEQ ID No.1, the reverse primer comprises a nucleotide sequence as shown in SEQ ID No.2, and the TXZT probe comprises a nucleotide sequence as shown in SEQ ID No.3.
[0009] The TXZT specific primer pair with nucleotide sequences as shown in SEQ ID No.1 and SEQ ID No.2 and the TXZT probe with nucleotide sequence as shown in SEQ ID No.3 provided in the present application can perform specific and highly sensitive PCR amplification and detection on the DNA of Pterocarpus santalinus, thereby enabling rapid, accurate and less destructive detection and identification of whether the wood to be tested is Pterocarpus santalinus.
[0010] In some embodiments of the present application, the 5' end of the TXZT probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.
[0011] The present application labels the TXZT probe with a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end, so that the TXZT probe has high specificity and sensitivity in the detection of DNA of Pterocarpus santalinus.
[0012] In some embodiments of the present application, the fluorescent reporter group is selected from FAM, CY3, CY5, ROX or VIC.
[0013] In some embodiments of the present application, the fluorescence quenching group is selected from BHQ1, BHQ2, BHQ3 or MGB.
[0014] The present application selects suitable fluorescent reporter groups and fluorescent quencher groups to label the probe, thereby performing specific and highly sensitive detection of the DNA of Pterocarpus santalinus.
[0015] In some embodiments of the present application, the target gene of the TXZT specific primer pair includes the nucleotide sequence shown in SEQ ID No.4.
[0016] The present application selects a target gene whose nucleotide sequence is shown in SEQ ID No. 4, and designs the above-mentioned TXZT specific primer pair and TXZT probe based on the target gene to perform specific and highly sensitive PCR amplification and detection of the DNA of Pterocarpus santalinus.
[0017] In a second aspect, an embodiment of the present application provides a kit for identifying Pterocarpus santalinus, comprising the primer probe set provided in the first aspect.
[0018] The kit for identifying Pterocarpus santalinus provided in the present application includes the above-mentioned primer probe set, which can perform specific and highly sensitive PCR amplification and detection on the DNA of Pterocarpus santalinus, thereby being able to quickly, accurately, and less destructively detect and identify whether the wood to be tested is Pterocarpus santalinus.
[0019] In some embodiments of the present application, the kit further comprises a PCR reaction solution, and the PCR reaction solution comprises dNTP, DNA polymerase and PCR buffer.
[0020] In some embodiments of the present application, the PCR reaction solution includes: based on a reaction system of 20-30 μL, a forward primer concentration of 0.1-0.7 μM, a reverse primer concentration of 0.1-0.7 μM, and a TXZT probe concentration of 0.3-1.5 μM.
[0021] The present application sets forward primers, reverse primers and probes of appropriate concentrations in the PCR reaction solution, and can perform specific and highly sensitive PCR amplification and detection on the DNA of Pterocarpus santalinus.
[0022] In a third aspect, the present application provides an identification method for Pterocarpus santalinus, comprising:
[0023] (1) Extracting DNA from the wood sample to be tested;
[0024] (2) using the kit provided by the second aspect to perform fluorescent PCR detection using the DNA in the wood sample to be tested extracted in step (1) as a template;
[0025] (3) Determine whether the wood to be tested is red sandalwood based on the results of fluorescent PCR testing.
[0026] The identification method of Pterocarpus santalinus provided in the present application first extracts DNA from the wood sample to be tested, and then uses the kit including the TXZT specific primer pair and the TXZT probe to perform fluorescent PCR detection using the extracted DNA from the wood sample to be tested as a template, which can quickly, accurately, and less destructively detect and identify whether the wood to be tested is Pterocarpus santalinus.
[0027] In some embodiments of the present application, 0.1-2 g of the wood sample to be tested is taken for detection and identification.
[0028] The present application uses an appropriate amount of wood samples to be tested for testing and identification, and can quickly and accurately detect and identify the results, thereby causing less damage to the wood.
[0029] In some embodiments of the present application, the fluorescent PCR amplification reaction conditions are: pre-denaturation reaction at 92-98°C for 2 minutes; denaturation at 93-97°C for 14-16 seconds, annealing at 55°C for 25-35 seconds, extension at 70-74°C for 20-25 seconds, 40 cycles; final extension at 72°C for 7-8 minutes.
[0030] The present application adopts appropriate fluorescent PCR amplification reaction conditions for amplification detection, which can perform rapid and accurate detection with specificity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.
[0032] Figure 1This is a diagram of the DNA extraction results of the ZT1 and ZT2 samples provided in Experimental Example 1 of the present application.
[0033] Figure 2 This is a graph of the fluorescence PCR amplification detection results of the ZT1 and ZT2 samples provided in Experimental Example 1 of the present application.
[0034] Figure 3 This is a graph of the fluorescence PCR amplification detection results of the ZT3 sample provided in Experimental Example 2 of this application. DETAILED DESCRIPTION
[0035] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0036] At present, the traditional method of identifying red sandalwood has problems such as long inspection time, high misjudgment rate, destructiveness to the wood and large amount of wood used.
[0037] To this end, the embodiment of the present application provides a primer-probe set for identifying Pterocarpus santalinus, comprising a TXZT-specific primer pair and a TXZT probe, the TXZT-specific primer pair comprising a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence as shown in SEQ ID No.1, the reverse primer comprises a nucleotide sequence as shown in SEQ ID No.2, and the TXZT probe comprises a nucleotide sequence as shown in SEQ ID No.3.
[0038] The TXZT specific primer pair with nucleotide sequences as shown in SEQ ID No.1 and SEQ ID No.2 and the TXZT probe with nucleotide sequence as shown in SEQ ID No.3 provided in the present application can perform specific and highly sensitive PCR amplification and detection on the DNA of Pterocarpus santalinus, thereby enabling rapid, accurate and less destructive detection and identification of whether the wood to be tested is Pterocarpus santalinus.
[0039] In some embodiments of the present application, the 5' end of the TXZT probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group. The 5' end of the TXZT probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group, so that the TXZT probe has high specificity and sensitivity in the detection of DNA of Pterocarpus santalinus.
[0040] As an example, the fluorescent reporter group can be selected from, but not limited to, FAM, CY3, CY5, ROX or VIC. The fluorescent quencher group can be selected from, but not limited to, BHQ1, BHQ2, BHQ3 or MGB. The probe is labeled with a suitable fluorescent reporter group and a fluorescent quencher group, so as to perform specific and highly sensitive detection of the DNA of Pterocarpus santalinus.
[0041] In some embodiments of the present application, the target gene of the TXZT specific primer pair includes the nucleotide sequence shown in SEQ ID No. 4. A target gene having a nucleotide sequence as shown in SEQ ID No. 4 is selected, and the above-mentioned TXZT specific primer pair and TXZT probe are designed based on the target gene to perform specific and highly sensitive PCR amplification and detection of the DNA of Pterocarpus santalinus.
[0042] The embodiment of the present application provides a kit for identifying Pterocarpus santalinus, comprising the primer probe set as described above.
[0043] Since the kit for identifying Pterocarpus santalinus includes the above-mentioned primer probe group, the above-mentioned primer probe group can perform specific and highly sensitive PCR amplification and detection on the DNA of Pterocarpus santalinus, so that the kit including the primer probe group can also perform specific and highly sensitive PCR amplification and detection on the DNA of Pterocarpus santalinus, thereby quickly, accurately and less destructively detecting whether the wood to be tested is Pterocarpus santalinus.
[0044] In some embodiments of the present application, the kit further includes a PCR reaction solution, which includes dNTP, DNA polymerase and PCR buffer. As an example, the PCR reaction solution includes Premix Ex Taq reagent (Takara); the Premix Ex Taq solvent includes 1.25 U of Ex Taq DNA polymerase, 200 μM single dNTP and 2 mM MgCl2. The PCR reaction solution may also include other commercially available Premix Ex Taq reagents.
[0045] It should be noted that the primer probe set is added to the PCR reaction solution so that the PCR reaction solution includes the primer probe set.
[0046] In some embodiments of the present application, the PCR reaction solution also includes a DNA template.
[0047] In some embodiments of the present application, the PCR reaction solution includes: based on a reaction system of 20-30 μL, a forward primer concentration of 0.1-0.7 μM, a reverse primer concentration of 0.1-0.7 μM, and a TXZT probe concentration of 0.3-1.5 μM. As an example, the concentration of the forward primer can be, but is not limited to, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM; the concentration of the reverse primer can be, but is not limited to, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM; the concentration of the TXZT probe can be, but is not limited to, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM. By setting appropriate concentrations of the forward primer, reverse primer and probe in the PCR reaction solution, specific and highly sensitive PCR amplification and detection of the DNA of Pterocarpus santalinus can be performed.
[0048] In some embodiments of the present application, the kit also includes a sample DNA extraction reagent, a positive control substance, a negative control substance, and a blank control substance. As an example, the sample DNA extraction reagent can adopt Plant Genomic DNA Kit (TIANGEN), or the sample DNA extraction reagent identical to the Plant Genomic DNA Kit can be directly prepared.
[0049] The present application embodiment provides a method for identifying red sandalwood, comprising:
[0050] (1) Extracting DNA from the wood sample to be tested;
[0051] (2) using the above-mentioned kit to perform fluorescent PCR detection using the DNA in the wood sample to be tested extracted in step (1) as a template;
[0052] (3) Determine whether the wood to be tested is red sandalwood based on the results of fluorescent PCR testing.
[0053] The identification method of Pterocarpus santalinus of the present application first extracts DNA from the wood sample to be tested, and then uses the kit including the TXZT specific primer pair and the TXZT probe to perform fluorescent PCR detection using the extracted DNA from the wood sample to be tested as a template, which can quickly, accurately, and less destructively detect and identify whether the wood to be tested is Pterocarpus santalinus.
[0054] In some embodiments of the present application, 0.1-2g of the wood sample to be tested is taken for detection and identification. As an example, but not limited to, 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, 1.0g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 2.0g of the wood sample to be tested is taken for detection and identification. Using an appropriate amount of the wood sample to be tested for detection and identification can quickly and accurately detect and identify the results, thereby less destructive to the wood.
[0055] In some embodiments of the present application, the fluorescent PCR amplification reaction conditions are: 92-98°C pre-denaturation reaction for 2 minutes; 93-97°C denaturation for 14-16 seconds, 55°C annealing for 25-35 seconds, 70-74°C extension for 20-25 seconds, 40 cycles; 72°C final extension for 7-8 minutes. The use of appropriate fluorescent PCR amplification reaction conditions for amplification detection can be performed quickly and accurately with specificity and high sensitivity. As an example, the fluorescent PCR amplification reaction conditions are: 95°C pre-denaturation reaction for 2 minutes; 95°C denaturation for 15 seconds, 55°C annealing for 30 seconds, 72°C extension for 20 seconds, 40 cycles; 72°C final extension for 7 minutes.
[0056] The features and performance of the present application are further described in detail below in conjunction with the embodiments.
[0057] Example 1
[0058] Determination of target gene sequence:
[0059] According to the known gene sequence of Pteroearpus dalbergioides Benth., a gene sequence with good specificity and suitable fragment length is selected as the target sequence for detection. After homology comparison and retrieval, the selected target sequence is determined to be a DNA sequence with high specificity.
[0060] The specific detection target gene sequence obtained is shown in SEQ ID No.4: AGTTTATTTTGA AATAATTCATTTTTTTTTCCTTACAATACAAAGAGAAATNTATGTTTGGCAAAATTTTGAAAAAGGGGGTAAAATNTTCAATGTNTTTCTTTAGATNTTAAAAAGGTAAAAAAGATAAAAAAAGGGGGGGTAAGATATATGGGTAATTAATCAAAGAAGAATTAGTTTTCCTTTTGAGAACCGAAGGAAGGTTTTTCCCCTGACCCGGAGCAATG.
[0061] Example 2
[0062] Design and synthesis of primers and probes:
[0063] According to the determined target gene sequence and the design principles of primers and probes, the primers and probes are designed as follows:
[0064] The forward primer TXZT Primer-F is shown in SEQ ID No. 1: 5′-GGGGATAAGATA TATGGCTAATT-3′;
[0065] The reverse primer TXZT Primer-R is shown in SEQ ID No. 2: 5′-TGGAACTGCCA TCCAACTAGTA-3′;
[0066] The probe TXZT Primer Probe is shown in SEQ ID No. 3: 5'-FAM-TGTCTTTCACATGACCTGTAGCAATG-BHQ1-3'.
[0067] The designed primers and probes were compared for homology on NCBI, and the results showed that the primer and probe sequences were only 100% similar to the reported Pterocarpus santalinus sequences and the determined target gene sequences. In addition, the comparison results showed that although the primer and probe sequences had certain similarities with other tree species, they could not effectively combine with the sequences of other tree species.
[0068] Therefore, the primers and probes designed above are specific to Pterocarpus santalinus DNA, and can only detect Pterocarpus santalinus during PRC amplification.
[0069] Example 3
[0070] This embodiment provides a kit for identifying Pterocarpus santalinus, comprising a sample DNA extraction reagent, a PCR reaction solution, a positive control substance, a negative control substance, and a blank control substance. Among them, the sample DNA extraction reagent uses Plant Genomic DNA Kit (TIANGEN); the positive control substance is: a plasmid solution containing a specific amplified fragment of Pterocarpus santalinus; the negative control substance is: a plasmid solution without a specific amplified fragment of Pterocarpus santalinus; the blank control substance is: physiological saline or ddH2O without any substance added. The PCR reaction solution calculated based on a reaction system of 25 μL is shown in Table 1.
[0071] Table 1 PCR reaction solution
[0072]
[0073]
[0074] The sequences of primers and probes are:
[0075] TXZT Primer-F:5'-GGGGATAAGATATATGGCTAATT-3';
[0076] TXZT Primer-R:5'-TGGAACTGCCATCCAACTAGTA-3';
[0077] TXZT Primer Probe:5'-FAM-TGTCTTTCACATGACCTGTAGCAATG-BHQ 1-3'.
[0078] Test Example 1
[0079] In this test example, the kit for identifying red sandalwood provided in Example 3 was used to perform real-time fluorescence PCR detection on red sandalwood and red sandalwood, as follows:
[0080] (1) Sample pretreatment: Two bracelets were selected, and 0.5 g of samples were taken from the holes of the bracelets using a spoon. They were numbered ZT1 (No. 07231, collection of China Red Sandalwood Museum, from the Telangana region of Andhra Pradesh, India) and ZT2 (Dye Red Sandalwood from Zambia). ZT1 and ZT2 samples were ground into uniform powder using a mortar and 2000 mesh quartz sand to maximize the surface area for DNA release;
[0081] (2) DNA extraction from wood samples: Lysis buffer was added to the ground ZT1 and ZT2 powder samples to rupture the cell membrane and release DNA. Column chromatography was used to purify the DNA to remove proteins, polysaccharides, and other impurities that may interfere with the PCR reaction. The genomic DNA of the ZT1 and ZT2 samples was extracted using the Plant Genomic DNA Kit (TIANGEN) according to the instructions.
[0082] (3) Endogenous gene detection: The quality and purity of the DNA extracted from the two groups of samples ZT1 and ZT2 were detected using the plant endogenous gene tRNALeu and the kit for identifying Pterocarpus santalinus provided in Example 3; wherein the primer and probe sequences (standard) of tRNALeu were:
[0083] The forward primer tRNALeu-F is shown in SEQ ID No. 5: 5'-CGAAATCGGTAGAC GCTACG-3';
[0084] The reverse primer tRNALeu-R is shown in SEQ ID No.6: 5'-TTCCATTGAGTCTCT GCACCT-3';
[0085] The probe tRNALeu Probe is shown in SEQ ID No.7: 5'-FAM-GCAATCCTGAG CCAAATCC-TAMIRA-3';
[0086] The results are as follows Figure 1 As shown, DNA from samples ZT1 and ZT2 was successfully extracted, and the quality of the DNA was verified by the detection of the endogenous gene tRNALeu;
[0087] (4) Real-time fluorescence PCR amplification and detection: The DNA samples of ZT1 and ZT2 extracted and verified in steps (2)-(3) were used as templates, respectively, and the kit for identifying Pterocarpus santalinus provided in Example 3 was used for fluorescence PCR amplification detection; the fluorescence PCR amplification reaction conditions were: 95°C pre-denaturation reaction for 2 min; 95°C denaturation for 15 s, 55°C annealing for 30 s, 72°C extension for 20 s, 40 cycles; 72°C final extension for 7 min;
[0088] The results are as follows Figure 2 As shown, the ZT1 sample showed the expected amplification curve and Ct value, confirming that it was Pterocarpus santalinus; while the ZT2 sample showed no amplification, excluding the possibility that it was Pterocarpus santalinus; the kit for identifying Pterocarpus santalinus provided by the present application can specifically amplify the DNA of the Pterocarpus santalinus ZT1 sample; at the same time, the dye Dalbergia truncatum ZT2 sample of the same genus was negative and could not be amplified; this indicates that the kit for identifying Pterocarpus santalinus provided by the present application, including the primer probe set provided in Example 2, has high specificity for the DNA of Pterocarpus santalinus.
[0089] Test Example 2
[0090] In this test example, the kit for identifying Pterocarpus santalinus provided in Example 3 was used to perform real-time fluorescence PCR detection on Pterocarpus santalinus to test the sensitivity, as follows:
[0091] The sandalwood bracelet beads from the China Red Sandalwood Museum's collection No. 07113 were selected, with the experimental number ZT3 (core material of sandalwood from Xinlin, Mysore, India). They were first dried to ensure the accuracy of the weight; graded sampling was performed according to the amounts of 0.1g, 0.5g, 1.0g and 2.0g to ensure the accuracy of each sample; the treatment and real-time fluorescence PCR amplification and detection were performed in the same manner as in Experimental Example 1; the Ct value of each sample was recorded, that is, the number of cycles to reach the fluorescence threshold, and the DNA yield extracted from each sample was measured to ensure the accuracy and repeatability of the data. The results are shown in FIG. Figure 3 shown.
[0092] Depend on Figure 3 The results show that as the sample size increases, the yield of extracted xylem genomic DNA increases linearly, while the Ct value decreases proportionally, indicating that the kit for identifying Pterocarpus santalinus provided by the present application, including the primer probe set provided in Example 2, has high sensitivity to the DNA of Pterocarpus santalinus.
[0093] At the same time, the experimental results also show that even when the sample size is only 0.1g, DNA information can be successfully detected, indicating that the kit for identifying red sandalwood provided by the present application, including the primer probe set provided in Example 2, has extremely high sensitivity to the DNA of red sandalwood. A relatively small amount of sample can also be detected with high sensitivity, which causes less damage to the identification of wood.
[0094] Since the detection requires a small amount of samples and does not rely on the morphological and structural integrity of the wood, the kit for identifying red sandalwood provided in the present application, including the primer probe group provided in Example 2, can be used not only to detect red sandalwood wood with complete morphology, but also to identify red sandalwood finished products or semi-finished products such as bracelets, handles, ornaments, and even incense powder, which are difficult to identify in their original form.
[0095] In summary, the TXZT specific primer pair with nucleotide sequences as shown in SEQ ID No.1 and SEQ ID No.2 and the TXZT probe with nucleotide sequence as shown in SEQ ID No.3 provided in the present application can perform specific and highly sensitive PCR amplification and detection on the DNA of Pterocarpus santalinus, so that the kit including the primer probe set can also perform specific and highly sensitive PCR amplification and detection on the DNA of Pterocarpus santalinus, thereby quickly, accurately and less destructively detecting whether the wood to be tested is Pterocarpus santalinus.
[0096] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.
Claims
1. A primer probe set for identifying Pterocarpus santalinus, characterized in that: It comprises a TXZT specific primer pair and a TXZT probe, wherein the TXZT specific primer pair comprises a forward primer and a reverse primer; the forward primer comprises a nucleotide sequence as shown in SEQ ID No.1, the reverse primer comprises a nucleotide sequence as shown in SEQ ID No.2, and the TXZT probe comprises a nucleotide sequence as shown in SEQ ID No.
3.
2. The primer probe set according to claim 1, characterized in that: The 5' end of the TXZT probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.
3. The primer probe set according to claim 2, characterized in that: The fluorescent reporter group is selected from FAM, CY3, CY5, ROX or VIC; And / or, the fluorescence quenching group is selected from BHQ1, BHQ2, BHQ3 or MGB.
4. The primer probe set according to any one of claims 1 to 3, characterized in that: The target gene of the TXZT specific primer pair includes the nucleotide sequence shown in SEQ ID No.
4.
5. A kit for identifying red sandalwood, characterized in that: Comprising the primer probe set according to any one of claims 1 to 4.
6. The kit according to claim 5, characterized in that The kit also includes a PCR reaction solution, which includes dNTPs, DNA polymerase and PCR buffer.
7. The kit according to claim 6, characterized in that The PCR reaction solution comprises: based on a reaction system of 20-30 μL, the concentration of the forward primer is 0.1-0.7 μM, the concentration of the reverse primer is 0.1-0.7 μM, and the concentration of the TXZT probe is 0.3-1.5 μM.
8. A method for identifying red sandalwood, characterized in that: include: (1) Extracting DNA from the wood sample to be tested; (2) using the kit according to any one of claims 5 to 7 to perform fluorescent PCR detection using the DNA extracted from the wood sample to be tested in step (1) as a template; (3) Determine whether the wood to be tested is red sandalwood based on the fluorescence PCR test results.
9. The identification method according to claim 8, characterized in that: Take 0.1-2g of the wood sample to be tested for detection and identification.
10. The identification method according to claim 8, characterized in that: The fluorescent PCR amplification reaction conditions are as follows: pre-denaturation reaction at 92-98°C for 2 min; denaturation at 93-97°C for 14-16 s, annealing at 55°C for 25-35 s, extension at 70-74°C for 20-25 s, 40 cycles; and final extension at 72°C for 7-8 min.