Kit for detecting natural musk and application and method thereof

Abstract

The invention provides a kit for detecting natural musk as well as application and a method thereof. The kit for detecting the natural musk comprises an RPA (recombinase polymerase amplification) primer pair, a colloidal gold labeled detection probe and a lateral flow chromatography test strip, when the kit provided by the invention is used for detecting a to-be-detected sample, firstly, genome DNA of the to-be-detected sample is extracted, and room-temperature RPA amplification is performed on the genome DNA of the to-be-detected sample by adopting the RPA amplification primer pair, so that an amplification product is obtained; and secondly, mixing the amplification product with a colloidal gold labeled detection probe, dropwise adding the mixture on a lateral flow chromatography test strip, and judging whether the natural musk is contained in the sample to be detected or not according to a strip of the lateral flow chromatography test strip. The kit provided by the invention not only has the advantages of simplicity and convenience in operation, strong anti-interference capability, high sensitivity, strong specificity and the like, but also can realize on-site nondestructive detection, and is convenient for inspectors to master and operate.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a kit for detecting natural musk and an application and method thereof. Background Art

[0002] Musk refers to a dry aromatic substance secreted from the scent glands between the navel and genitals of mature male musk deer. It has a unique fragrance with deep, mellow and lasting characteristics and is often used in high-end perfumes and cosmetics. At the same time, musk is also a precious animal medicinal material, which has the effects of opening the mind, refreshing the mind, promoting blood circulation, relieving swelling and pain, and has a certain therapeutic effect on central nervous system, respiratory and cardiovascular system diseases. It also has a strong anti-inflammatory effect. Due to the small number of wild musk deer and the poor effect of artificial breeding, musk is one of the precious natural spices. However, due to the difficulty of extracting musk, its price is relatively expensive, and many artificial musks have appeared on the market. Therefore, how to identify natural musk is one of the hot topics in this field.

[0003] At present, the commonly used identification techniques include chemical analysis technology, spectroscopy technology and DNA identification technology based on polymerase chain reaction (PCR). Chemical analysis technology is one of the most commonly used methods for musk identification. Chemical analysis technology can distinguish natural musk from artificial musk by analyzing the characteristics of chemical components in musk. Commonly used analysis methods include high-performance gas chromatography-mass spectrometry, high-performance liquid chromatography, gas chromatography, etc. These technologies have high precision and high sensitivity, but require a long analysis time and complex operation procedures and equipment. Spectroscopic techniques such as Fourier transform infrared spectroscopy (FTIR) mainly use the absorption spectrum and fluorescence spectrum of musk and the optical properties of musk samples to detect and identify musk. Compared with traditional empirical detection methods, this method has the advantages of rapidity, sensitivity and non-destructiveness. However, the spectra of natural musk and artificial musk are very similar, which is not conducive to the intuitive identification of natural musk and artificial musk.

[0004] Recombinase Polymerase Amplification (RPA) has the advantages of simple operation and low equipment requirements. Compared with traditional nucleic acid amplification methods such as PCR, RPA reaction does not require professional operation and does not rely on large and expensive imported instruments. In addition, the reaction temperature of RPA technology is between 25-42°C, so it is very simple to use RPA reaction, and it can even be directly incubated in the human armpit, which is particularly suitable for rapid on-site detection. How to achieve high sensitivity, high specificity, and high stability of musk identification based on RPA technology has become one of the new focuses of attention in this field. Summary of the invention

[0005] The invention provides a kit for detecting natural musk, comprising RPA primers for amplifying natural musk genomic DNA, a detection probe labeled with colloidal gold and a lateral flow chromatography test strip.

[0006] The present invention also provides a method for detecting natural musk and distinguishing natural musk from artificial musk, which is carried out using the above kit. Compared with the commonly used PCR method, the method provided by the present invention does not require an instrument and can complete the reaction at room temperature. Compared with the color development reaction of the test strip using antigen and antibody, this method reduces the material cost by means of the double-stranded complementary principle, and can realize simple, fast and low-cost on-site detection.

[0007] In a first aspect, the present invention provides a kit for detecting natural musk, comprising an RPA amplification primer pair, a colloidal gold-labeled detection probe, and a lateral flow chromatography test strip, wherein: The RPA amplification primer pair includes a first primer having a structure as shown in Formula 1 and a second primer having a structure as shown in Formula 2: 5'-X-(n)a-3'Formula 1 In Formula 1, X is the first labeling group, (n)a is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 1; 5'-(n)bH-(n)c-3' Formula 2 In Formula 2, (n)b is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 2, H is a spacer group, and (n)c is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 3; The nucleotide sequence of the detection probe is SEQ ID NO: 4; The lateral flow chromatography test strip comprises a first layer and a second layer arranged on the surface of the first layer, wherein the second layer comprises a sample absorption pad, a nitrocellulose membrane and a water absorption pad, the nitrocellulose membrane is provided with a detection line and a quality control line, an antibody against a first labeling group is fixed on the detection line, and a complex formed by an antibody against the first labeling group and a quality control probe is fixed on the quality control line, and the structure of the quality control probe is shown in Formula 3: 5'-X-(n)b-3' Formula 3 In Formula 3, X is the first labeling group, and (n)b is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 2.

[0008] In the kit as described above, the first labeling group is biotin, and the antibody against the first labeling group is streptavidin.

[0009] In the above-mentioned kit, the spacer group is C3 spacer, and the specific structure is -CH2CH2CH2-.

[0010] In the above-mentioned kit, the nucleotide sequence of SEQ ID NO: 1 is specifically as follows: 5'-TTCTGATTYTTTGGHCACCCRGAA-3'.

[0011] In the above-mentioned kit, the nucleotide sequence of SEQ ID NO: 2 is specifically as follows: 5′-TTGGTCGTGGTGGTGGTTT-3′.

[0012] In the above-mentioned kit, the nucleotide sequence of SEQ ID NO:3 is specifically as follows: 5'-TAAAYATATGGTGGGCTCATAC-3'.

[0013] In the above-mentioned kit, the nucleotide sequence of SEQ ID NO:4 is specifically as follows: 5'-AAACCACCACCACGACCAATTTTTTTTTTTTTTTT-3'.

[0014] The kit as described above can modify the single-stranded DNA molecule whose nucleotide sequence is SEQ ID NO: 4 with thiol to obtain a detection probe, and mix the detection probe with colloidal gold to obtain a detection probe labeled with colloidal gold. The thiol modification can be carried out according to conventional technical means in the art, and the detection probe labeled with colloidal gold can be prepared by the following preparation method: placing the colloidal gold solution and the detection probe solution under light-proof conditions for 16-24 hours, adding phosphate buffer and NaCl solution to the above-mentioned mixed system, continuing to place for 8-12 hours, then centrifuging to remove the supernatant, collecting the precipitate and resuspending it with phosphate buffer and NaCl solution to obtain the detection probe labeled with colloidal gold. Furthermore, the particle size of the nano-gold in the colloidal gold solution is 25 nm, and the concentration of the detection probe in the detection probe solution is 100 μM.

[0015] Furthermore, the concentration of the phosphate buffer is 10 mM, the volume ratio of the detection probe solution to the phosphate buffer is 1:2.8, and the concentration of the NaCl solution is 2M.

[0016] The kit as described above can be sprayed on the nitrocellulose membrane with a solution containing an antibody against the first labeling group and a solution containing the antibody against the first labeling group and a quality control probe, respectively, and allowed to stand for 1-2 hours and dry to obtain a nitrocellulose membrane with a detection line and a quality control line.

[0017] In the kit as described above, the concentration of the antibody against the first labeling group in the solution containing the antibody against the first labeling group used to form the test line can be 5 mg / mL. Further, the concentration of streptavidin in the solution containing streptavidin used to form the test line can be 5 mg / mL.

[0018] In the kit as described above, when forming the quality control line, the solution containing the antibody against the first labeling group and the quality control probe is used, the concentration of the antibody against the first labeling group can be 1 mg / mL, and the concentration of the quality control probe can be 100 μM.

[0019] The kit as described above further comprises a reaction buffer solution. Further, the reaction buffer solution can be a TE buffer solution, which is used to mix with the amplification product and the detection probe labeled with colloidal gold, and the mixed solution is dripped onto the sample absorption pad of the lateral flow chromatography test strip.

[0020] In a second aspect, the present invention provides the use of any of the above-mentioned kits in the following aspects: C1) Application in identifying natural musk and artificial musk; C2) Application in the preparation of products for distinguishing natural musk from artificial musk; C3) Application in detecting whether a sample contains natural musk; C4) Application in preparing a product for detecting whether a sample contains natural musk.

[0021] In a third aspect, the present invention provides a method for distinguishing natural musk from artificial musk, which is performed using any of the above-mentioned kits, comprising: Extracting genomic DNA samples of musk to be tested; Using the genomic DNA sample as a template, amplification is performed using an RPA amplification primer pair to obtain an amplification product; The amplification product and the detection probe labeled with colloidal gold are mixed to obtain a mixture, and the mixture is applied to the sample absorption pad of the lateral flow chromatography test strip, and the mixture is observed. When red strips appear on the detection line and the quality control line of the lateral flow chromatography test strip, it indicates that the musk to be detected is natural musk. When no red strips appear on the detection line but a red strip appears on the quality control line, it indicates that the musk to be detected is artificial musk. When no red strips appear on the detection line and the quality control line, it indicates that the test result is invalid.

[0022] In a fourth aspect, the present invention provides a method for detecting whether a sample contains natural musk, using any of the above-mentioned kits, comprising: Extracting genomic DNA from samples to be tested; Using the genomic DNA sample as a template, amplification is performed using an RPA amplification primer pair to obtain an amplification product; The amplification product and the detection probe labeled with colloidal gold are mixed to obtain a mixture, and the mixture is applied to the sample absorption pad of the lateral flow chromatography test strip, and the mixture is observed. When red strips appear on the detection line and the quality control line of the lateral flow chromatography test strip, it indicates that the sample to be detected contains natural musk. When no red strips appear on the detection line but red strips appear on the quality control line, it indicates that the sample to be detected does not contain natural musk. When no red strips appear on the detection line or the quality control line, it indicates that the test result is invalid.

[0023] In a fifth aspect, the present invention provides a DNA molecule selected from any one of A1) to A4): A1) the nucleotide sequence is a single-stranded DNA of SEQ ID NO: 1; A2) the nucleotide sequence is a single-stranded DNA of SEQ ID NO: 2; A3) the nucleotide sequence is a single-stranded DNA of SEQ ID NO: 3; A4) The nucleotide sequence is a single-stranded DNA of SEQ ID NO:4.

[0024] In a sixth aspect, the present invention provides a chemical substance, wherein the structure of the chemical substance is selected from any one of B1) to B3): B1) 5'-X-(n)a-3' Formula 1 In Formula 1, X is the first labeling group, (n)a is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 1; B2) 5'-(n)bH-(n)c-3' Formula 2 In Formula 2, (n)b is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 2, H is a spacer group, and (n)c is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 3; B3) 5'-X-(n)b-3' Formula 3 In Formula 3, X is the first labeling group, and (n)b is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 2.

[0025] In a seventh aspect, the present invention provides the use of the above-mentioned DNA molecules or chemical substances in the following aspects: C1) Application in identifying natural musk and artificial musk; C2) Application in the preparation of products for distinguishing natural musk from artificial musk; C3) Application in detecting whether a sample contains natural musk; C4) Application in preparing a product for detecting whether a sample contains natural musk.

[0026] The invention provides a kit for detecting natural musk, comprising specific primers for amplifying natural musk, a detection probe labeled with colloidal gold and a lateral flow chromatography test strip, which not only realizes the detection of natural musk and the identification of natural musk and artificial musk, but also uses the double-strand complementary principle to carry out a color development reaction, which is helpful to reduce material costs compared with the color development reaction of a test strip using an antigen and antibody.

[0027] The present invention realizes the detection of natural musk and the identification of natural musk and artificial musk based on recombinase polymerase amplification (RPA) technology and lateral flow chromatography test strips. Compared with the commonly used PCR method, the method provided by the present invention does not require any instrument, the reaction can be completed at room temperature, the reaction time is only 20 minutes, and the result can be interpreted by the naked eye in no more than 3 minutes. It has the advantages of simple operation, strong anti-interference ability, high sensitivity, strong specificity, etc., can realize simple, fast, low-cost on-site non-destructive detection, and is convenient for inspectors to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The RPA detection result diagram of the RPA amplification primer pair provided by the present invention on natural musk, artificial musk genomic DNA samples and water; wherein M represents marker, 1 and 2 represent natural musk, 3 represents artificial musk, and 4 represents water; Figure 2 A schematic diagram of the natural musk detection principle provided by an embodiment of the present invention; Figure 3 This is a graph showing the specificity experimental results of the detection method provided by the present invention; wherein 1-2 is natural musk, 3 is artificial musk, and 4 is water; Figure 4 The sensitivity test result diagram of the detection method provided by the present invention; wherein 1-8 are respectively the extraction solution of natural musk genomic DNA diluted to 2 ng / μL, 1 ng / μL, 5×10 -1 ng / μL, 10 -1 ng / μL、5×10 -2 ng / μL、3×10 -2 ng / μL, 10 -2 ng / µL, 0ng / μL, a total of 8 concentration gradients. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0030] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0031] The natural musk used in the following examples was purchased from Zhejiang Tongjuntang Chinese Medicine Co., Ltd., and the artificial musk was distributed by China Aba Prefecture Pharmaceutical Corporation. All samples were stored at -20°C before use; streptavidin, sodium chloride, phosphate buffer, and TE buffer were all purchased from Beijing Bainovi Biotechnology Co., Ltd.; primers / probes were synthesized by Shanghai Shenggong Bioengineering Technology Co., Ltd.

[0032] Example 1: Design of specific primers for detecting natural musk 1. Extraction of genomic DNA samples from natural musk First, two natural musk samples and one artificial musk sample were cut into pieces and placed in 0.5 ml microcentrifuge tubes respectively. The genomic DNA samples of the musk samples were extracted using the Tiangen Trace DNA Extraction Kit. The concentration and purity of the genomic DNA samples were tested using the Nanodrop 2000c Nucleic Acid Protein Analyzer, and the A260nm / A280nm value was calculated to determine whether the extracted DNA met the experimental requirements. The results showed that the DNA extracted from the three musk samples met the experimental requirements, and their A260nm / A280nm values ​​were between 1.8 and 2.0.

[0033] 2. Primer design and establishment of RPA system Genetic informatics technology was used to analyze and search for the COI gene of the natural musk genome, and specific primers as shown in Table 1 were designed with the COI gene of natural musk as the target gene. The primers described in Table 1 were synthesized in the Shanghai Biotechnology Laboratory and stored at -20°C. The primers were diluted to a final concentration of 10 μM with sterile deionized water and stored at -4°C for future use.

[0034] Table 1 Primer sequence information

[0035] In Table 1, Y represents C or T, H represents A, C or T, and R represents A or G.

[0036] The natural musk and artificial musk genomic DNA samples obtained in the above steps were used as templates, and the RPA reaction was performed using the TwistAmp® DNA amplification kit. The reaction was carried out in a constant temperature water bath, and ddH2O was used as a negative control. The total volume of the reaction mixture for each RPA reaction was 50 μL, including: 1.2 μL forward primer (10 μM), 1.2 μL reverse primer (10 μM), 2 μL DNA template, 41.5 μL buffer A, 1.6 μL ddH2O, and 2.5 μL buffer B. After the reaction mixture was reacted at 40°C for 20 minutes, the amplification product was detected by 3% agarose gel electrophoresis (AGE).

[0037] Test results such as Figure 1 As shown, it can be seen that the PCR amplification product of natural musk is positive, showing as a band at 249bp, while the detection results of artificial musk and ddH2O are negative, verifying that the above PCR primers are feasible.

[0038] Example 2, Detection of lateral flow chromatography test strips 1. Principle description The detection principle of the lateral flow chromatography test strip provided in this embodiment is as follows Figure 2 As shown, first, the musk genomic DNA to be detected is used as a template, and the modified forward primer and reverse primer are used to perform RPA amplification on the musk genomic DNA to be detected to obtain an amplified product; secondly, the amplified product is mixed with a detection probe labeled with colloidal gold, and a partial nucleotide sequence of the detection probe is complementary to a partial nucleotide sequence of the reverse primer to form a complex; finally, when the mixed solution of the amplified product and the detection probe labeled with colloidal gold is dripped onto the sample pad of the lateral flow chromatography test strip, the mixed solution flows to the nitrocellulose membrane through capillary action, and when the mixed solution flows to the detection line, if the target amplified product exists, the target amplified product forms a complex with the detection probe labeled with colloidal gold, and the biotin labeled at the 5' end of the forward primer is combined with the streptavidin fixed on the detection line, that is, the colloidal gold stays at the detection line to produce a red strip. The excess colloidal gold-labeled detection probe continues to be chromatographed, and when it flows to the quality control line, it is complementary to the quality control probe on the quality control line, so that the quality control line produces a red strip. When the target amplification product does not exist, the colloidal gold cannot stay at the detection line, but complementarily pairs with the quality control probe sequence on the quality control line, and the quality control line still produces a red strip.

[0039] Table 2 Substances required for lateral flow chromatography test strips name Sequence (5'-3') 5' end modification 3' end modification Quality control probe (SEQ ID NO:2) TTGGTCGTGGTGGTGGTTT Biotin / Forward primer (SEQ ID NO: 1) TTCTGATTYTTTGGHCACCCRGAA Biotin / Reverse primer (SEQ ID NO:2-3) TTGGTCGTGGTGGTGGTTT / iSpC3 / TAAAYATATGGTGGGCTCATAC / / Detection probe (SEQ ID NO: 4) AAACCACCACCACGACCAATTTTTTTTTTTTTTTT / HS-SH C6 In Table 2, Y represents C or T, H represents A or C or T, and R represents A or G. The 5' end of the forward primer is connected to biotin (represented by Biotin); the nucleotide sequence of the second primer consists of SEQ ID NO: 2-3, and 3 methylene groups are modified between SEQ ID NO: 2 and SEQ ID NO: 3 (represented by iSpC3); the 3' end of the detection probe is connected to the HS-SH C6 group for connection to colloidal gold; the 5' end of the quality control probe is connected to biotin (represented by Biotin).

[0040] 2. Detection of lateral flow chromatography test strips 2.1. Preparation of lateral flow chromatography test strips Streptavidin was prepared into a streptavidin solution at a concentration of 5 mg / mL, and the streptavidin solution was sprayed on a nitrocellulose membrane to form a test line (Test Line); after 1 mg / mL of streptavidin was reacted with 100 μM of quality control probe at room temperature for two hours, the mixed solution was sprayed on the nitrocellulose membrane to form a quality control line (Control Line), thereby obtaining a nitrocellulose membrane coated with a test line and a quality control line.

[0041] The lateral flow chromatography test strip was prepared by pasting a sample absorption pad (Shanghai Jieyi Biotechnology Co., Ltd., JY-BX111), the nitrocellulose membrane coated with the detection line and the quality control line prepared above, and a water-absorbing pad on a bottom plate in sequence.

[0042] 2.2 Preparation of colloidal gold labeled detection probe Place 500μl of gold nanoparticles (25nm in diameter) and 20μL of 100μM detection probe in a dark place for 16h, add 56μL of 10mM phosphate buffer, add 2M NaCl to 0.3M, and place for 8h. Centrifuge at 4℃, 16100×g for 30min, and remove the supernatant. Finally, resuspend the precipitate in 0.3M NaCl and 10mM phosphate buffer and place at 4℃ for later use.

[0043] 2.3. A method for rapidly identifying natural musk and artificial musk using the above-mentioned kit comprises the following steps: 1) Preparation of DNA template: Extraction was performed according to the instructions of the whole genome nucleic acid extraction kit to finally obtain 100 μL of DNA extract.

[0044] 2) RPA amplification reaction: Use the genomic DNA extract obtained in step 1) to amplify using the RPA kit and detect the amplified product by electrophoresis. The RPA reaction system is: 41.5 μL of reaction buffer, 2 μL of forward primer and 2 μL of reverse primer, 2 μL of DNA template, and 2.5 μL of Buffer B to start the reaction. Reaction process: 37°C for 20 min.

[0045] 3) Purification of target fragment: Use a purification kit to purify the product.

[0046] 4) Sample detection: Lay the test strip flat, take 4 μL of amplification product, 10 μL of colloidal gold-labeled detection probe and 50 μL of TE buffer, mix them and drop them into the sample absorption pad of the test strip. Interpret the results at room temperature. If the test line T is colored, the result is positive; if the test line T is not colored, the result is negative; if the quality control line C is not colored, the test strip is invalid.

[0047] 2.4. Specificity experiment: According to the established reaction system, a specificity experiment was conducted on 2 natural musks, 1 artificial musk and a blank template. The test results are as follows Figure 3 As shown, except for natural musk, artificial musk and blank template had no bands, proving good specificity.

[0048] 2.5. Sensitivity experiment: In order to determine the minimum detection amount of this kit, the extract containing natural musk genomic DNA was diluted to 2 ng / μL, 1 ng / μL, 5×10 -1 ng / μL, 10 -1 ng / μL、5×10 -2 ng / μL、3×10 -2 ng / μL, 10 -2 ng / μL, 0 for a total of 8 concentration gradients, and the test was carried out according to the above method. The test results are as follows Figure 4 As shown, it can be seen that the minimum detection limit of the present invention is 3×10 -2 ng / μL.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for detecting natural musk, characterized in that: It includes an RPA amplification primer pair, a colloidal gold-labeled detection probe, and a lateral flow chromatography test strip, wherein: The RPA amplification primer pair includes a first primer having a structure as shown in Formula 1 and a second primer having a structure as shown in Formula 2: 5'-X-(n)a-3' Formula 1 In Formula 1, X is the first labeling group, (n)a is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 1; 5'-(n)bH-(n)c-3' Formula 2 In Formula 2, (n)b is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 2, H is a spacer group, and (n)c is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 3; The nucleotide sequence of the detection probe is SEQ ID NO: 4; The lateral flow chromatography test strip comprises a first layer and a second layer arranged on the surface of the first layer, wherein the second layer comprises a sample absorption pad, a nitrocellulose membrane and a water absorption pad, the nitrocellulose membrane is provided with a detection line and a quality control line, an antibody against a first labeling group is fixed on the detection line, and a complex formed by an antibody against the first labeling group and a quality control probe is fixed on the quality control line, and the structure of the quality control probe is shown in Formula 3: 5'-X-(n)b-3' Formula 3 In Formula 3, X is the first labeling group, and (n)b is a single-stranded DNA having a nucleotide sequence of SEQ ID NO:

2.

2. The kit according to claim 1, characterized in that The first labeling group is biotin, and the antibody against the first labeling group is streptavidin.

3. The kit according to claim 1, characterized in that The spacer group is -CH2CH2CH2-.

4. The kit according to any one of claims 1 to 3, characterized in that The kit also includes a reaction buffer solution.

5. Use of the kit according to any one of claims 1 to 4 in the following aspects: C1) Application in identifying natural musk and artificial musk; C2) Application in the preparation of products for distinguishing natural musk from artificial musk; C3) Application in detecting whether a sample contains natural musk; C4) Application in preparing a product for detecting whether a sample contains natural musk.

6. A method for distinguishing natural musk from artificial musk, characterized in that: The method is carried out using the kit according to any one of claims 1 to 4, comprising: Extracting genomic DNA samples of musk to be tested; Using the genomic DNA sample as a template, amplification is performed using an RPA amplification primer pair to obtain an amplification product; The amplification product and the detection probe labeled with colloidal gold are mixed to obtain a mixture, and the mixture is applied to the sample absorption pad of the lateral flow chromatography test strip, and the mixture is observed. When red strips appear on the detection line and the quality control line of the lateral flow chromatography test strip, it indicates that the musk to be detected is natural musk. When no red strips appear on the detection line but a red strip appears on the quality control line, it indicates that the musk to be detected is artificial musk. When no red strips appear on the detection line and the quality control line, it indicates that the test result is invalid.

7. A method for detecting whether a sample contains natural musk, characterized in that: The method is carried out using the kit according to any one of claims 1 to 4, comprising: Extracting genomic DNA from samples to be tested; Using the genomic DNA sample as a template, amplification is performed using an RPA amplification primer pair to obtain an amplification product; The amplification product and the detection probe labeled with colloidal gold are mixed to obtain a mixture, and the mixture is applied to the sample absorption pad of the lateral flow chromatography test strip, and the mixture is observed. When red strips appear on the detection line and the quality control line of the lateral flow chromatography test strip, it indicates that the sample to be detected contains natural musk. When no red strips appear on the detection line but red strips appear on the quality control line, it indicates that the sample to be detected does not contain natural musk. When no red strips appear on the detection line or the quality control line, it indicates that the test result is invalid.

8. A DNA molecule, characterized in that It is selected from any one of A1) to A4): A1) the nucleotide sequence is a single-stranded DNA of SEQ ID NO: 1; A2) the nucleotide sequence is a single-stranded DNA of SEQ ID NO: 2; A3) the nucleotide sequence is a single-stranded DNA of SEQ ID NO: 3; A4) The nucleotide sequence is a single-stranded DNA of SEQ ID NO:

4.

9. A chemical substance, characterized in that The structure of the chemical substance is selected from any one of B1) to B3): B1) 5'-X-(n)a-3' Formula 1 In Formula 1, X is the first labeling group, (n)a is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 1; B2) 5'-(n)bH-(n)c-3' Formula 2 In Formula 2, (n)b is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 2, H is a spacer group, and (n)c is a single-stranded DNA having a nucleotide sequence of SEQ ID NO: 3; B3) 5'-X-(n)b-3' Formula 3 In Formula 3, X is the first labeling group, and (n)b is a single-stranded DNA having a nucleotide sequence of SEQ ID NO:

2.

10. Use of the DNA molecule according to claim 8 or the chemical substance according to claim 9 in the following aspects: C1) Application in identifying natural musk and artificial musk; C2) Application in the preparation of products for distinguishing natural musk from artificial musk; C3) Application in detecting whether a sample contains natural musk; C4) Application in preparing a product for detecting whether a sample contains natural musk.

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