Specific molecular marker combination, primer probe group and kit for identifying artificial cordyceps sinensis and wild cordyceps sinensis in tibet autonomous region and application thereof

Abstract

The application discloses a specific molecular marker combination, a primer probe group, a kit and application thereof for identifying artificial Ophiocordyceps sinensis and wild Ophiocordyceps sinensis in the Tibet Autonomous Region, and the molecular marker combination comprises SNP1, SNP2 and SNP5; the SNP1 is located at the 339bp position of the nucleotide sequence shown in SEQ ID NO:1, and the polymorphism is C or T; the SNP2 is located at the 354bp position of the nucleotide sequence shown in SEQ ID NO:1, and the polymorphism is A or C; and the SNP5 is located at the 398bp position of the nucleotide sequence shown in SEQ ID NO:1, and the polymorphism is G or T. The molecular marker combination of the application scheme can be effectively used for distinguishing the artificial Ophiocordyceps sinensis and the wild Ophiocordyceps sinensis in the Tibet Autonomous Region.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to specific molecular marker combinations, primer and probe sets, reagent kits, and their applications for identifying artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis from the Tibet Autonomous Region. Background Technology

[0002] Cordyceps sinensis is a complex of the fruiting body and larval remains of a fungus belonging to the Clavicipitaceae family, which parasitizes the larvae of insects in the Hepialidae family of Lepidoptera. It was first recorded in the *Ben Cao Cong Xin* (New Compilation of Materia Medica). Modern medical research has confirmed that the secondary metabolites contained in Cordyceps sinensis (such as nucleoside compounds, polysaccharides, and sterols) can act on the human immune, cardiovascular, respiratory, genitourinary, endocrine, and nervous systems. Pharmacologically, it not only treats liver and kidney damage and lung inflammation but also possesses various effects such as anti-oxidation, anti-fatigue, and anti-tumor activity.

[0003] Cordyceps sinensis generally grows only in high-altitude areas above 3000 meters. The scarcity of wild resources and the huge market demand lead to high prices for commercially available Cordyceps sinensis, resulting in numerous counterfeit products. Driven by high prices, unscrupulous merchants employ various methods to fake its quality, such as using toothpick-like herbs, sulfur-treated herbs, and 3D-printed herbs, leading to inconsistent product quality in the market and frequent instances of misleading and deceiving consumers, seriously hindering the healthy and orderly development of the industry. Meanwhile, driven by profit and demand, and thanks to the efforts of many scientists, artificially cultivated Cordyceps sinensis has emerged. However, in terms of nutritional components and medicinal effects, wild Cordyceps sinensis contains richer nutrients and active substances. Furthermore, research shows that the proportion of inorganic arsenic in artificially cultivated Cordyceps sinensis is much higher than in wild Cordyceps sinensis, while the proportion of organic arsenic is significantly lower, and inorganic arsenic is indeed much more toxic than organic arsenic. Inorganic arsenic, commonly known as "arsenic trioxide," was officially classified as a human carcinogen by the International Agency for Research on Cancer in 1980. Ingesting large amounts in a short period can cause acute poisoning, while long-term excessive intake can damage the skin and cause chronic liver disease. Currently, the successful cultivation of artificially produced Cordyceps sinensis is consistent with wild Cordyceps sinensis in terms of insect body, fungal strain, appearance, and microstructure. Traditional methods (empirical methods based on morphology, color, and odor) are no longer sufficient to distinguish between wild and artificial Cordyceps sinensis, necessitating a scientific and effective method for identification.

[0004] In recent years, researchers have integrated spectroscopy (such as near-infrared spectroscopy), chemical analysis (such as HPLC-MS), imaging (3D structured light scanning), and genetics (DNA barcoding) to achieve comprehensive tracing from morphology to the molecular level. However, these technologies are expensive, have long experimental cycles, high technical barriers, rely on specialized equipment and interdisciplinary talents, and are difficult to popularize. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a combination of molecular markers for identifying artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis from the Tibet Autonomous Region.

[0006] The present invention also proposes a primer-probe set for detecting the above-mentioned molecular marker combinations.

[0007] This invention also proposes the application of the above-mentioned molecular marker combinations and primer-probe sets.

[0008] The present invention also proposes a kit having the above-described primer-probe combination.

[0009] The present invention also proposes applications of the above-mentioned reagent kit.

[0010] This invention also proposes a method for distinguishing between artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis from the Tibet Autonomous Region.

[0011] According to one aspect of the present invention, a molecular marker combination for identifying artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis of Tibet Autonomous Region is proposed, comprising SNP1, SNP2 and SNP5; wherein SNP1 is located at position 339 bp of the nucleotide sequence shown in SEQ ID NO:1, and has a polymorphism of C or T; wherein SNP2 is located at position 354 bp of the nucleotide sequence shown in SEQ ID NO:1, and has a polymorphism of A or C; and wherein SNP5 is located at position 398 bp of the nucleotide sequence shown in SEQ ID NO:1, and has a polymorphism of G or T.

[0012] According to a second aspect of the invention, a primer-probe set for detecting the above-described molecular marker combination is provided.

[0013] In some embodiments of the present invention, the primer-probe set includes a primer set and a fluorescent probe; The primer set includes a forward primer with the sequence shown in SEQ ID NO:2 and a reverse primer with the sequence shown in SEQ ID NO:3; The sequence of the fluorescent probe is shown in SEQ ID NO:4.

[0014] In some embodiments of the present invention, the fluorescent probe is labeled with a fluorescent reporter gene and a fluorescent quencher gene at both ends.

[0015] In some embodiments of the present invention, the fluorescent probe is labeled with a fluorescent reporter gene at its 5' end and a fluorescent quencher gene at its 3' end.

[0016] In some embodiments of the present invention, the fluorescent reporter group includes one of 6-FAM, HEX, JOE, CY3, CY5, VIC, ROX, and Texas Red.

[0017] In some embodiments of the present invention, the fluorescence quenching group includes one of Dabcyl, BHQ1, BHQ2, Eclipse, and MGB.

[0018] In a third aspect of the invention, the application of the above-described molecular marker combination and primer-probe set in any of the following is proposed: (1) Identify wild and artificial Cordyceps sinensis in Tibet Autonomous Region; (2) Prepare products for identifying wild and artificial Cordyceps sinensis in Tibet Autonomous Region; (3) Testing for wild Cordyceps sinensis in Tibet Autonomous Region; (4) Prepare products for testing wild Cordyceps sinensis in Tibet Autonomous Region; (5) Detection of artificially cultivated Cordyceps sinensis; (6) Prepare products for testing artificial Cordyceps sinensis.

[0019] According to a fourth aspect of the present invention, a kit is provided containing the aforementioned primer and probe set.

[0020] In some embodiments of the present invention, the kit further includes a method for detecting... ITS A primer and probe set for detecting genes. ITS The primer sequences in the gene primer and probe set are shown in SEQ ID NO:5 and SEQ ID NO:6; the probe sequence is shown in SEQ ID NO:7.

[0021] In some embodiments of the present invention, the probe sequence is labeled with a fluorescent reporter gene and a fluorescent quencher gene at both ends.

[0022] In some embodiments of the present invention, the method for detection ITS The fluorescent groups of the probe sequences in the primer-probe set of the gene are different from those in the primer-probe set of the molecular marker combination used to detect and distinguish between artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis of Tibet Autonomous Region.

[0023] In some embodiments of the present invention, the 5' end of the probe sequence is labeled with a fluorescent reporter gene and the 3' end is labeled with a fluorescent quencher gene.

[0024] In some embodiments of the present invention, the fluorescent reporter group includes one of 6-FAM, HEX, JOE, CY3, CY5, VIC, ROX, and Texas Red.

[0025] In some embodiments of the present invention, the fluorescence quenching group includes one of Dabcyl, BHQ1, BHQ2, Eclipse, and MGB.

[0026] In some embodiments of the present invention, the kit further includes PCR buffer, dNTPs, MgCl2, and Taq enzyme. The present invention provides a standardized Cordyceps sinensis identification kit by combining a primer-probe set for detecting molecular marker combinations with reagents such as enzymes and buffers required for fluorescent PCR reactions, thus providing a convenient and unified tool for the detection of Cordyceps sinensis.

[0027] In a fifth aspect of the invention, the use of the kit in any of the following is proposed: 1) Identify wild and cultivated Cordyceps sinensis in Tibet Autonomous Region; 2) Prepare products for identifying wild and artificially cultivated Cordyceps sinensis in the Tibet Autonomous Region; 3) Testing for wild Cordyceps sinensis in the Tibet Autonomous Region; 4) Prepare products for testing wild Cordyceps sinensis in the Tibet Autonomous Region; 5) Detection of artificially cultivated Cordyceps sinensis; 6) Prepare products for detecting artificially cultivated Cordyceps sinensis; 7) Identifying genuine Cordyceps sinensis; 8) Prepare products for identifying the authenticity of Cordyceps sinensis.

[0028] In some embodiments of the present invention, the identification of the authenticity of Cordyceps sinensis is achieved through the following steps: using a detection method... ITS The primer and probe set for the gene is used to detect the DNA in the sample to be tested.

[0029] In some embodiments of the present invention, the method for detection ITS The primer sequences in the gene primer and probe set are shown in SEQ ID NO:5 and SEQ ID NO:6; the probe sequence is shown in SEQ ID NO:7.

[0030] In a sixth aspect of the present invention, a method for identifying artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis of the Tibet Autonomous Region is proposed. The detection method includes the following steps: using the above-mentioned molecular marker combination, primer probe set or kit to detect the DNA of the sample to be tested.

[0031] In some embodiments of the present invention, the sample to be tested includes the insect body portion of a Cordyceps sinensis sample. The present invention extracts DNA from the insect body portion of the Cordyceps sinensis sample to be tested (insect DNA is more stable than that of the fruiting body and is more closely associated with the host insect's genes), and performs fluorescent PCR detection using the aforementioned primer-probe combination or kit; by analyzing the strength of the amplification signal of specific molecular markers in the detection results, it directly determines whether the sample is wild Cordyceps sinensis from the Tibet Autonomous Region, thereby achieving a clear distinction between artificial and wild varieties.

[0032] In some embodiments of the present invention, when the polymorphic site of SNP1 of the molecular marker combination is C, the polymorphic site of SNP2 is A, and the polymorphic site of SNP5 is G, it indicates that the sample is wild Cordyceps sinensis from Tibet Autonomous Region; when the polymorphism of SNP1 is T, the polymorphism of SNP2 is C, and the polymorphic site of SNP5 is T, it indicates that the sample is artificially cultivated Cordyceps sinensis.

[0033] In some embodiments of the present invention, the detection is performed using fluorescent PCR.

[0034] In some embodiments of the present invention, the detection system of the fluorescent PCR is: PCR buffer with a final concentration of 1×, upstream primer with a final concentration of 0.05-0.5 μmol / L, downstream primer with a final concentration of 0.05-0.5 μmol / L, probe with a final concentration of 0.05-0.5 μmol / L, dNTPs with a final concentration of 0.1-0.3 mmol / L, Taq enzyme with a final concentration of 0.2-0.5 U / μL, and MgCl2 with a final concentration of 1-3 mmol / L.

[0035] In some embodiments of the present invention, the amplification program of the fluorescent PCR is as follows: pre-denaturation at 94-96°C for 1.5-2.5 min; then entering the cycling stage: denaturation at 94-96°C for 8-12 s, annealing at 58-62°C, extension and fluorescence data acquisition for 25-35 s, 40-50 cycles.

[0036] In some embodiments of the present invention, the amplification program of the real-time fluorescence PCR is as follows: pre-denaturation at 95°C for 2 min; then entering the cycling stage: denaturation at 95°C for 10 s, annealing at 60°C, extension and fluorescence data acquisition for 30 s, for 45 cycles.

[0037] In some embodiments of the present invention, after detection using fluorescent PCR, the method further includes a step of statistically analyzing the Ct values ​​of the samples and interpreting the results; the result interpretation is as follows: When FAM fluorescence is detected and the Ct value is ≤36, it indicates that wild Cordyceps sinensis from Tibet Autonomous Region is detected in the sample, and the sample to be tested is wild Cordyceps sinensis from Tibet Autonomous Region. When FAM fluorescence is detected and the Ct value is >36, it indicates that artificial Cordyceps sinensis is detected in the sample, and the sample to be tested is artificial Cordyceps sinensis.

[0038] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The molecular marker combination of the present invention uses the relevant genes of the ghost moth (the host insect of Cordyceps sinensis) in the main producing area of ​​wild Cordyceps sinensis in Tibet Autonomous Region as the research object, and through gene sequence comparison, differential analysis and other means, screens and determines specific molecular markers that are unique to wild Cordyceps sinensis in Tibet Autonomous Region. The molecular markers are located in... Cytb Genetic differentiation is the core basis for distinguishing between wild and artificial varieties. It can eliminate interference from artificial Cordyceps sinensis and Cordyceps sinensis from other regions, and can be effectively used to differentiate between artificial Cordyceps sinensis and wild Cordyceps sinensis from Tibet Autonomous Region.

[0039] A fluorescent PCR primer-probe set was designed based on the specific molecular marker combination of wild Cordyceps sinensis selected from the Tibet Autonomous Region. This ensures that the primers and probes effectively bind to the target molecular markers of wild Cordyceps sinensis and perform specific amplification. It can be effectively used to distinguish between artificial Cordyceps sinensis and wild Cordyceps sinensis from the Tibet Autonomous Region, ensuring detection specificity from the source and achieving high detection accuracy.

[0040] This invention, based on fluorescent PCR technology and equipped with dedicated primers and probes, enables single-sample detection within 3-4 hours and supports multi-channel batch detection, improving efficiency by 3-5 times compared to traditional methods. Furthermore, while existing technologies often rely on whole-strain samples or complex pretreatment, this invention only requires a portion of the insect's DNA (insect DNA is more stable, easier to extract, and requires less sample volume), thus reducing sample loss. Attached Figure Description

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a homology comparison analysis diagram of the sequence of the host insect *Hepialus fasciatus* from Baqing County, Nagqu City, Tibet Autonomous Region (the main producing area of ​​*Cordyceps sinensis*) and the sequence of the host insect *Hepialus fasciatus* from artificially produced *Cordyceps sinensis* in Embodiment 1 of this invention. Figure 2 As described in the embodiments of the present invention Cytb The results of the detection of wild Cordyceps sinensis in Tibet Autonomous Region using gene primer and probe sets are shown in the figure. Figure 3 As described in the embodiments of the present invention Cytb The detection results of the gene primer and probe set on artificial Cordyceps sinensis; Figure 4 As described in the embodiments of the present invention Cytb ROC curves of gene primer and probe sets for distinguishing between wild and cultivated Cordyceps sinensis in Tibet Autonomous Region. Detailed Implementation

[0042] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0043] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0044] Example 1: Specific molecular markers for identifying artificial and wild Cordyceps sinensis from Tibet Autonomous Region (1) Screening of molecular markers Insect mitochondrial DNA (mtDNA) is characterized by its simple structure, absence of spacers and introns, lack of repetitive sequences, strict maternal inheritance, and near-absence of recombination. Using mtDNA to reveal the phylogenetic relationships among different insect groups has become a hot topic in current insect molecular systematics research. Among these, cytochrome b (…) Cytb Located in the phospholipid bilayer of the inner mitochondrial membrane, it is one of the 13 protein-coding genes in mitochondria whose structure and function are best understood. Its moderate evolutionary rate makes it suitable for studying phylogenetic relationships at the species level, and it is considered one of the most reliable molecular markers for resolving classification and phylogenetic problems. Therefore, it was selected... Cytb The gene was used as a target gene for screening specific SNP sites. The sequence of the host insect *Hepialus chinensis* (gene accession number: AF124304.1) from Baqing County, Nagqu City, Tibet Autonomous Region (a major producing area of ​​*Cordyceps sinensis*), was compared with the sequence of the artificially cultivated host insect *Hepialus chinensis* (gene accession number: NC_028348.1) using DNAMAN software. This screening revealed the specific SNP sites in *Cordyceps sinensis* from the main producing area of ​​Tibet Autonomous Region. Cytb Five specific SNP molecular marker sites on the gene (sequence homology alignment analysis diagram as shown) Figure 1 As shown), SNP1 is located in Cytb At position 339bp of the gene, the polymorphism is C or T; SNP2 is located at... Cytb At position 354bp of the gene, the polymorphism is A or C; SNP3 is located at... Cytb At position 384bp of the gene, the polymorphism is either G or A; SNP4 is located at... Cytb At position 387bp of the gene, the polymorphism is G or T; SNP5 is located at... CytbAt position 398bp of the gene, the polymorphism is either G or T.

[0045] Through further screening, this embodiment of the invention selects SNP1, SNP2, and SNP5 to distinguish between artificial and wild Cordyceps sinensis from Tibet Autonomous Region. Specifically, when SNP1 is C, SNP2 is A, and SNP5 is G, the sample is wild Cordyceps sinensis from Tibet Autonomous Region. When SNP1 is T, SNP2 is C, and SNP5 is T, the sample is artificial Cordyceps sinensis.

[0046] Cytb The gene sequence is as follows: (SEQ IDNO:1).

[0047] (2) Screening and design of primer and probe sequences Analysis was conducted on the unique SNP molecular marker sites of wild Cordyceps sinensis from the Tibet Autonomous Region. Based on the primer and probe design principles of fluorescent PCR, SNP1 was ultimately selected. Cytb The 339th bp position of the gene (CT) and SNP2 ( Cytb Specific primer and probe sequences were designed at the 354bp (AC) and SNP5 (398bp (GT)) sites to accurately identify and match unique sites in wild Cordyceps sinensis from the Tibet Autonomous Region. Simultaneously, targeting the specific sites of Cordyceps sinensis... ITS Primers and probes were designed from conserved gene regions to identify whether a sample is genuine Cordyceps sinensis and to monitor the effectiveness of the extraction process, thus screening for the best quality samples. Cytb Gene primer and probe set. This invention provides a method for identifying artificially cultivated and wild Cordyceps sinensis from the Tibet Autonomous Region, and for distinguishing genuine from counterfeit Cordyceps sinensis. Cytb Primer probe set, used for detection ITS The sequences of the primer and probe set are shown in Table 1.

[0048] Table 1

[0049] Among them, the underlined nucleotides are polymorphic sites.

[0050] Table 2

[0051] Table 3

[0052] Using the nucleic acid sequences of 32 wild and cultivated Cordyceps sinensis samples from the Tibet Autonomous Region as templates, quantitative real-time PCR was performed using the system shown in Table 2 and the procedure shown in Table 3. The detection results are as follows: Figure 2-3 As shown in the figure, Cytb The gene primer and probe set can effectively distinguish between wild and cultivated Cordyceps sinensis in the Tibet Autonomous Region; therefore, it was selected... Cytb The gene primer and probe set was used for subsequent experiments.

[0053] Example 2: A kit for detecting and identifying artificial and wild Cordyceps sinensis from Tibet Autonomous Region. This embodiment provides a kit for detecting and identifying artificially cultivated and wild Cordyceps sinensis from the Tibet Autonomous Region. The kit contains PCR buffer (purchased from Nanjing Novizan Biotechnology Co., Ltd.), primers and / or probes, MgCl2, Taq enzyme, and sterile purified water. Common PCR buffers consist of buffer systems such as Tris-HCl, dNTPs, MgCl2, KCl, Triton X-100, and other components required for the catalytic reaction.

[0054] The reaction system used for real-time PCR is shown in Table 2 above, and the reaction procedure is shown in Table 3. Generally, the total volume of a single PCR reaction tube is 20-200µL. Appropriate concentrations of primers and / or probes, Taq enzyme, etc. are added to the PCR buffer. The concentration of primers and / or probes can be between 0.05µM and 5µM.

[0055] The method of using the kit is as follows: 1. Extraction of insect DNA Nucleic acid was extracted from Cordyceps sinensis samples using the following extraction method: Take an appropriate weight of Cordyceps sinensis insect body sample and perform nucleic acid extraction according to the instructions of the Tiangen Plant Genomic DNA Extraction Kit (catalog number: DP305): (1) Take about 30mg of the insect body part of Cordyceps sinensis, add liquid nitrogen and grind it thoroughly; (2) Quickly transfer the ground powder into a centrifuge tube containing 700 µL of 65℃ preheated buffer GP1 and quickly invert to mix. Place the centrifuge tube in a 37℃ oven for 30 min, add 20 µL of proteinase K, and place the centrifuge tube in a 65℃ water bath for 2-3 h. Invert the centrifuge tube several times during the water bath process to mix the sample. (3) Add 700 µL of chloroform, mix thoroughly, and centrifuge at 12,000 rpm (~13,400×g) for 5 min; (4) Carefully transfer the upper aqueous phase obtained in the previous step into a new centrifuge tube, add 700 µL of buffer GP2, and mix thoroughly; (5) Transfer the mixed liquid into the adsorption column CB3, centrifuge at 12,000 rpm (~13,400 x g) for 30 sec, and discard the waste liquid; (6) Add 500 µL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400 x g) for 30 sec, discard the waste liquid, and put the adsorption column CB3 into the collection tube; (7) Add 600 µL of wash buffer PW to the adsorption column CB3, centrifuge at 12,000 rpm (~13,400 x g) for 30 sec, discard the waste liquid, and place the adsorption column CB3 into the collection tube. Repeat this step twice; (8) Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400 x g) for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material; (9) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200µL of elution buffer TE to the middle of the adsorption membrane, place at room temperature for 2-5 min, centrifuge at 12,000 rpm (~13,400xg) for 2 min, and collect the solution into the centrifuge tube.

[0056] 2. Sample detection (1) Reagent preparation: After equilibrating the test kit to room temperature, mix PCR reaction solution A (12.5 μL / T) and PCR reaction solution B (7.5 μL / T) according to the number of samples to be tested, vortex until homogeneous, and then briefly centrifuge before use.

[0057] (2) Sample addition: Add 5 μL of the processed sample to the corresponding 0.2 mL PCR reaction tube, then add 20 μL of the prepared detection reagent, cap the tube, shake well, and centrifuge at 3000 rpm for 30 seconds until there are no obvious bubbles in the liquid.

[0058] (3) PCR amplification: PCR amplification was performed on PCR instruments such as the SLAN-96S fully automated medical PCR analysis system by following the procedure shown in Table 3.

[0059] (4) Interpretation of test results: 1) If the Ct value of the HEX (VIC) channel of the sample is ≥45, it is determined to be fake Cordyceps sinensis; 2) If the sample shows a clear S-shaped amplification curve in the HEX (VIC) channel and the Ct value is <40, it is determined to be genuine Cordyceps sinensis. Then, the Ct value in the FAM channel is used to determine whether it is wild Cordyceps sinensis from the Tibet Autonomous Region. If the Ct value of the FAM channel is ≤36, it is determined to be wild Cordyceps sinensis from the Tibet Autonomous Region; If the Ct value of the FAM channel is greater than 36, it is determined to be artificial Cordyceps sinensis; 3) If the HEX (VIC) channel of the sample is 40≤Ct<45, it is necessary to resample and extract the sample for detection, and then interpret the sample results according to the above method.

[0060] Example 3: Analysis of the judgment values ​​of the composition of the present invention in distinguishing between wild and cultivated Cordyceps sinensis in Tibet Autonomous Region The kit prepared in Example 2 was used to distinguish between wild and artificially cultivated Cordyceps sinensis from another 32 known sources in Tibet Autonomous Region.

[0061] Table 4 shows the test data of Cordyceps sinensis samples using the composition of the present invention. Simultaneously, ROC curve fitting of the Ct values ​​was performed using data analysis software, and the results are as follows: Figure 4 As shown.

[0062] From Table 4 and Figure 4 As can be seen from this, when the cut-off value of Ct is set to 36, wild Cordyceps sinensis and artificial Cordyceps sinensis in Tibet Autonomous Region can be distinguished.

[0063] Table 4

[0064] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A molecular marker combination for identifying artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis from the Tibet Autonomous Region, characterized in that, The molecular marker combination includes SNP1, SNP2, and SNP5; SNP1 is located at position 339 bp of the nucleotide sequence shown in SEQ ID NO:1, with a polymorphism of C or T; SNP2 is located at position 354 bp of the nucleotide sequence shown in SEQ ID NO:1, with a polymorphism of A or C; and SNP5 is located at position 398 bp of the nucleotide sequence shown in SEQ ID NO:1, with a polymorphism of G or T.

2. A primer-probe set for detecting the molecular marker combination as described in claim 1.

3. The primer-probe set according to claim 2, characterized in that, The primer-probe set includes a primer set and a fluorescent probe; The primer set includes a forward primer with the sequence shown in SEQ ID NO:2 and a reverse primer with the sequence shown in SEQ ID NO:3; The sequence of the fluorescent probe is shown in SEQ ID NO:

4.

4. The primer-probe set according to claim 3, characterized in that, The fluorescent probe is labeled with a fluorescent reporter gene and a fluorescent quencher gene at its two ends, respectively; Preferably, the fluorescent reporter group includes one of 6-FAM, HEX, JOE, CY3, CY5, VIC, ROX, and Texas Red; Preferably, the fluorescence quenching group includes one of Dabcyl, BHQ1, BHQ2, Eclipse, and MGB.

5. The use of the molecular marker combination of claim 1 or the primer-probe set of any one of claims 2-4 in any of the following: (1) Identify wild and artificial Cordyceps sinensis in Tibet Autonomous Region; (2) Prepare products for identifying wild and artificial Cordyceps sinensis in Tibet Autonomous Region; (3) Testing for wild Cordyceps sinensis in Tibet Autonomous Region; (4) Prepare products for testing wild Cordyceps sinensis in Tibet Autonomous Region; (5) Detection of artificially cultivated Cordyceps sinensis; (6) Prepare products for testing artificial Cordyceps sinensis.

6. A reagent kit, characterized in that, The kit includes the primer and probe set as described in any one of claims 2-4.

7. The reagent kit according to claim 6, characterized in that, The kit also includes methods for detection ITS A primer and probe set for detecting genes. ITS The primer sequences in the gene primer and probe set are shown in SEQ ID NO:5 and SEQ ID NO:6; the probe sequences are shown in SEQ ID NO:

7.

8. The use of the kit according to claim 6 or 7 in any of the following: 1) Identify wild and cultivated Cordyceps sinensis in Tibet Autonomous Region; 2) Prepare products for identifying wild and artificially cultivated Cordyceps sinensis in the Tibet Autonomous Region; 3) Testing for wild Cordyceps sinensis in the Tibet Autonomous Region; 4) Prepare products for testing wild Cordyceps sinensis in the Tibet Autonomous Region; 5) Detection of artificially cultivated Cordyceps sinensis; 6) Prepare products for detecting artificially cultivated Cordyceps sinensis; 7) Identifying genuine Cordyceps sinensis; 8) Prepare products for identifying the authenticity of Cordyceps sinensis.

9. A method for distinguishing between artificially cultivated Cordyceps sinensis and wild Cordyceps sinensis from the Tibet Autonomous Region, characterized in that, The detection method includes the following steps: using the molecular marker combination as described in claim 1, the primer probe set as described in any one of claims 2-4, or the kit as described in claim 6 or 7 to detect the DNA of the sample to be tested; Preferably, the detection is performed using fluorescent PCR; More preferably, the detection system of the fluorescent PCR is: PCR buffer with a final concentration of 1×, upstream primer with a final concentration of 0.05-0.5 μmol / L, downstream primer with a final concentration of 0.05-0.5 μmol / L, probe with a final concentration of 0.05-0.5 μmol / L, dNTPs with a final concentration of 0.1-0.3 mmol / L, Taq enzyme with a final concentration of 0.2-0.5 U / μL, and MgCl2 with a final concentration of 1-3 mmol / L; More preferably, the amplification program of the fluorescent PCR is as follows: pre-denaturation at 94-96°C for 1.5-2.5 min; then entering the cycling stage: denaturation at 94-96°C for 8-12 s, annealing at 58-62°C, extension and fluorescence data acquisition for 25-35 s, 40-50 cycles.

10. The method according to claim 9, characterized in that, After detection using fluorescent PCR, the method further includes a step of statistically analyzing the Ct values ​​of the samples and interpreting the results; the result interpretation is as follows: When FAM fluorescence is detected and the Ct value is ≤36, it indicates that wild Cordyceps sinensis from Tibet Autonomous Region is detected in the sample, and the sample to be tested is wild Cordyceps sinensis from Tibet Autonomous Region. When FAM fluorescence is detected and the Ct value is >36, it indicates that artificial Cordyceps sinensis is detected in the sample, and the sample to be tested is artificial Cordyceps sinensis.

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