PCR primer pair and probe for identifying adulteration of saussurea involucrate and application of PCR primer pair and probe

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a PCR primer pair and a probe for identifying adulteration of saussurea involucrate and application of the PCR primer pair and the probe. Wherein the nucleotide sequence of the PCR primer pair is shown as SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequence of the probe is shown as SEQ ID NO: 3. Based on the PCR primer pair and / or probe for identifying adulteration of saussurea involucrata, provided by the invention, the adulteration condition of adulteration of saussurea involucrata and / or saussurea lancea in saussurea involucrata can be accurately and quickly identified.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and in particular relates to a PCR primer pair and a probe for adulteration identification of Saussurea involucrata and an application thereof. Background Art

[0002] Saussurea involucrata (Kar. et Kir.) Sch. Bip. is the dried aerial part of Saussurea involucrata (Kar. et Kir.) Sch. Bip., a plant of the genus Saussurea in the Asteraceae family. It has the effects of promoting blood circulation, nourishing the kidney, strengthening the bones and tendons, and removing wind and dampness. Saussurea involucrata grows in a very harsh environment and is a very precious medicinal material unique to the high mountainous areas in western my country. Due to its slow production speed and low natural reproduction capacity, as well as long-term excavation, its wild resources are on the verge of extinction. It has been listed as a national second-class protected plant. In addition, the Saussurea DC plant includes nearly 400 species, including S. involucrata (Kar. et Kir.) Sch. Bip., such as S. laniceps Hand.-Mazz. and S. medusa Maxim., most of which are distributed in the Qinghai-Tibet Plateau in my country.

[0003] Snow lotus is often called "snow lotus" in Tibet. In traditional Tibetan medicine, snow rabbit with jellyfish and snow rabbit with cotton head are also called medicinal "snow lotus". Due to the scarcity of wild snow lotus resources, the rich species of plants of the same genus, and the same local name, there are cases in the market where plants of the same genus such as snow rabbit with cotton head and snow rabbit with jellyfish are mixed with snow lotus. The current quality standard of snow lotus is simple and lacks specificity for species identification of Saussurea plants. Therefore, it is very necessary to establish an efficient, accurate and specific identification method for common and easily confused counterfeits of snow lotus.

[0004] Fluorescence quantitative PCR technology is currently the most commonly used component quantitative analysis method for gene expression level analysis, and has also been gradually applied in traditional Chinese medicine testing in recent years. Among them, the locked nucleic acid (LNA)-TaqMan probe is based on the TaqMan probe, and some bases in the probe are modified with LNA. By introducing LNA modification to increase the system Tm value, the specificity of the probe and the sensitivity of detection are greatly improved. At present, there is no relevant research on the application of LNA-TaqMan fluorescence quantitative PCR in the identification of Saussurea involucrata. Summary of the invention

[0005] The present invention develops a PCR primer pair and a probe for identifying adulteration of Saussurea tianshanica, and Saussurea tianshanica can be identified based on the primer pair and the probe.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On one hand, the present invention provides a PCR primer pair for identifying adulteration of Saussurea tianshanica, and the nucleotide sequences thereof are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0008] Another aspect of the present invention provides a PCR primer pair and a probe for identifying adulteration of Saussurea tianshanica. The nucleotide sequences of the PCR primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequence of the probe is shown in SEQ ID NO:3.

[0009] In another aspect, the present invention provides a detection reagent for identifying adulteration of Saussurea tianshanica, comprising the PCR primer pair for identifying adulteration of Saussurea tianshanica in the present invention or the PCR primer pair and probe for identifying adulteration of Saussurea tianshanica in the present invention.

[0010] In another aspect, the present invention provides a detection kit for identifying adulteration of Saussurea tianshanica, which comprises the detection reagent for identifying adulteration of Saussurea tianshanica of the present invention.

[0011] On the other hand, the present invention provides a PCR primer pair for identifying adulteration of Saussurea tianshanica in the present invention, or a PCR primer pair and a probe for identifying adulteration of Saussurea tianshanica in the present invention, used in the preparation of a detection product for identifying adulteration of Saussurea tianshanica.

[0012] On the other hand, the present invention provides a method for identifying adulterated snow rabbit jellyfish and / or snow rabbit cotton-head in Saussurea involucrata, comprising: (1) extracting DNA from a sample to be detected and a standard sample to obtain a DNA template of the sample to be detected and a DNA template of the standard sample; (2) using the PCR primer pair and probe for identifying adulteration of Saussurea involucrata in the present invention to perform PCR amplification on the DNA template of the sample to be detected and the DNA template of the standard sample to obtain a first CT value and a second CT value; (3) calculating the difference ΔCT between the first CT value and the second CT value, and identifying the content of adulterated snow rabbit jellyfish and / or snow rabbit cotton-head in Saussurea involucrata according to the absolute value of ΔCT.

[0013] Preferably, in the above identification method, when the absolute value of △CT is ≤4, the content of adulterated Snow Rabbit and / or Snow Rabbit in Tianshan Snow Lotus is ≥10%.

[0014] Preferably, in the above identification method, the PCR amplification program includes: 94°C / 3min, 94°C / 5sec, 62.5°C / 30sec, among which 94°C / 5sec, 62.5°C / 30sec.

[0015] The beneficial effects of the present invention at least include: based on the PCR primer pair and / or probe for identifying adulteration of Tianshan Saussurea involucrata provided by the present invention, adulterated jellyfish snow rabbit and / or cotton-headed snow rabbit in Tianshan Saussurea involucrata can be identified, and the adulteration of jellyfish snow rabbit and / or cotton-headed snow rabbit in Tianshan Saussurea involucrata can be accurately and quickly identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are the amplification curves for Snow Rabbit, Snow Rabbit, and Snow Lotus;

[0017] Figure 2 This is the sensitivity test amplification curve of the fluorescence quantitative PCR method of the present invention; the red curve is the DNA concentration of 0.1ng·μL –1 sample;

[0018] Figure 3 This is the specific amplification curve of the fluorescent quantitative PCR method for detecting jellyfish snow rabbits of the present invention, wherein the blue curve is the blank control;

[0019] Figure 4 This is the specific amplification curve of the fluorescent quantitative PCR method for detecting snow rabbits, wherein the blue curve is the blank control;

[0020] Figure 5 This is the specific amplification curve of detecting Saussurea involucrata by the fluorescent quantitative PCR method of the present invention, wherein the blue curve is the blank control. DETAILED DESCRIPTION

[0021] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0022] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0023] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0024] The embodiment of the present invention provides a PCR primer pair for identifying adulteration of Saussurea tianshanica, and the nucleotide sequences thereof are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0025] It should be noted that the PCR primer pair in the present invention can be used to identify adulteration of Saussurea involucrata with good specificity and high accuracy. Specifically, the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 are as follows:

[0026] TSXL4138-F(SEQ ID NO:1):5'-CGACACTCTTAATTGATACG-3';

[0027] TSXL4138-R (SEQ ID NO: 2): 5'-CGGTAAAGTTTGGAAGAC-3'.

[0028] The embodiment of the present invention also provides a PCR primer pair and a probe for identifying adulteration of Saussurea tianshanica. The nucleotide sequence of the PCR primer pair is shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequence of the probe is shown in SEQ ID NO:3.

[0029] It should be noted that the present invention can also perform fluorescent PCR identification based on PCR primer pairs and probes to more accurately identify the adulteration of Tianshan Saussurea involucrata. Specifically, the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are as follows:

[0030] TSXL4138-F(SEQ ID NO:1):5'-CGACACTCTTAATTGATACG-3';

[0031] TSXL4138-R(SEQ ID NO:1):5'-CGGTAAAGTTTGGAAGAC-3';

[0032] M4138-P2 (SEQ ID NO: 3): 5′-FAM-tttCcaTtcAttCcctttc-BHQ1-3′ (lower case letters indicate LNA modified bases).

[0033] The embodiment of the present invention also provides a detection reagent for identifying adulteration of Saussurea tianshanica, including the PCR primer pair for identifying adulteration of Saussurea tianshanica in the present invention or the PCR primer pair and probe for identifying adulteration of Saussurea tianshanica in the present invention.

[0034] It should be noted that the PCR primer pair or the PCR primer pair and the probe in the present invention can be mixed with a detection auxiliary reagent to prepare a detection reagent, and the detection auxiliary reagent includes a buffer solution and the like.

[0035] The embodiment of the present invention also provides a detection kit for identifying adulteration of Saussurea tianshanica, which comprises the detection reagent for identifying adulteration of Saussurea tianshanica in the present invention.

[0036] It should be noted that the detection reagent for identification of adulteration of Saussurea tussah in the present invention can be prepared into a detection kit, which is more convenient for transportation and use.

[0037] The embodiments of the present invention also provide a PCR primer pair for identifying adulteration of Saussurea tianshanica in the present invention or the use of the PCR primer pair and probe for identifying adulteration of Saussurea tianshanica in the present invention in the preparation of a detection product for identifying adulteration of Saussurea tianshanica.

[0038] The embodiment of the present invention also provides a method for identifying adulterated snow rabbits of jellyfish and / or snow rabbits of cotton head in Saussurea involucrata, comprising: (1) extracting DNA from a sample to be detected and a standard sample respectively to obtain a DNA template of the sample to be detected and a DNA template of the standard sample; (2) using the PCR primer pair and probe for identifying adulteration of Saussurea involucrata in the present invention to perform PCR amplification on the DNA template of the sample to be detected and the DNA template of the standard sample respectively to obtain a first CT value and a second CT value; (3) calculating the difference △CT between the first CT value and the second CT value, and identifying the content of adulterated snow rabbits of jellyfish and / or snow rabbits of cotton head in Saussurea involucrata according to the absolute value of △CT.

[0039] In some specific examples, in the above identification method, when the absolute value of △CT is ≤4, the content of adulterated jellyfish snow rabbit and / or cotton-head snow rabbit in Tianshan Snow Lotus is ≥10%.

[0040] In some specific examples, in the above identification method, the PCR amplification program includes: 94°C / 3min, 94°C / 5sec, 62.5°C / 30sec, among which 94°C / 5sec, 62.5°C / 30sec.

[0041] 1. Determination of primer pairs and probes

[0042] The chloroplast genome sequences of Saussurea tianshanica, Snow Rabbit, and Snow Rabbit were downloaded from the database of the National Center for Biotechnology Information (NCBI: https: / / www.ncbi.nlm.nih.gov / ). By comparing and analyzing the chloroplast genome sequences of the above three species, 141 single nucleotide polymorphism (SNP) sites shared by Snow Rabbit, Snow Rabbit, and Saussurea tianshanica were screened out with the sequence of Saussurea tianshanica (MH926106) as a reference. The best primer pairs for detection were designed using the proprietary algorithm using the Beacon Designer 8 software. Finally, the cytosine (C) > adenine (A) mutation at position 4138 was determined as the single nucleotide polymorphism (SNP) site, and specific probes and primer pairs for Snow Rabbit and Snow Rabbit were obtained. The probe (M4138-P2) was purified by high performance liquid chromatography (HPLC), and the 5′ end of the specific probe was labeled with the fluorescein group FAM, and the 3′ end was labeled with the non-fluorescein quencher group BHQ1. The probe and primer sequences are as follows:

[0043] TSXL4138-F:5'-CGACACTCTTAATTGATACG-3';

[0044] TSXL4138-R:5'-CGGTAAAGTTTGGAAGAC-3';

[0045] M4138-P2: 5'-FAM-tttCcaTtcAttCcctttc-BHQ1-3' (lowercase letters indicate LNA modified bases).

[0046] 2. PCR amplification detection

[0047] (I) Extraction of genomic DNA from samples of Snow Rabbit, Snow Rabbit and Snow Lotus

[0048] Take samples of Snow Rabbit of Jellyfish, Snow Rabbit of Cotton-head and Snow Lotus of Tianshan Mountains with accurate origin (for specific information, see Table 1), take 0.1g of each, wash them with 1ml of 75% ethanol and 1ml of sterilized ultrapure water respectively, absorb the surface moisture, and grind them in a ball mill (or grind them with liquid nitrogen) into fine powder; take 20mg of each, put them in a 1.5ml centrifuge tube, and use the Plant Genomic DNA Extraction Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract nucleic acid to obtain DNA template.

[0049] Table 1 Sample information table

[0050] Serial number Plant-based Latin name serial number 1 Tianshan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-01 2 Tianshan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-02 3 Tianshan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-03 4 Tianshan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-04 5 Tianshan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-05 6 Tianshan Snow Lotus Saussurea involucrata(Kar.et Kir.)Sch.Bip. TSXL-06 7 Jellyfish Snow Bunny S. medusa Maxim. SMXTZ-01 8 Jellyfish Snow Bunny S. medusa Maxim. SMXTZ-02 9 Jellyfish Snow Bunny S. medusa Maxim. SMXTZ-03 10 Jellyfish Snow Bunny S. medusa Maxim. SMXTZ-04 11 Jellyfish Snow Bunny S. medusa Maxim. SMXTZ-05 12 Jellyfish Snow Bunny S. medusa Maxim. SMXTZ-06 13 Jellyfish Snow Bunny S. medusa Maxim. SMXTZ-07 14 Snow rabbit S.laniceps Hand.-Mazz. MTXTZ-01 15 Snow rabbit S.laniceps Hand.-Mazz. MTXTZ-02 16 Snow rabbit S.laniceps Hand.-Mazz. MTXTZ-03 17 Snow rabbit S.laniceps Hand.-Mazz. MTXTZ-04 18 Snow rabbit S.laniceps Hand.-Mazz. MTXTZ-05 19 Snow rabbit S.laniceps Hand.-Mazz. MTXTZ-06 20 Snow rabbit S.laniceps Hand.-Mazz. MTXTZ-07

[0051] (II) Fluorescence PCR amplification reaction

[0052] The DNA template obtained above was added to the PCR reaction system for fluorescence PCR amplification reaction and CT value analysis, wherein the PCR reaction system is shown in Table 2 below.

[0053] Table 2 PCR reaction system

[0054]

[0055]

[0056] In addition, the PCR amplification reaction program was as follows: 94°C / 3 min, 94°C / 5 sec, 62.5°C / 30 sec, wherein 94°C / 5 sec, 62.5°C / 30 sec, the process was performed for 40 cycles.

[0057] (III) CT value analysis

[0058] The PCR amplification products were analyzed using a Bio-Rad CFX96 analyzer. The amplification curves of different samples are shown in Figure 1 As shown, the results show that the probe primers in the present invention were used to test different batches of Snow Rabbit and Snow Rabbit, and each obtained a positive result, while different batches of Snow Lotus in Tianshan Mountains all obtained negative results, further confirming that the primers and probes of the present invention can be used to identify adulterated Snow Rabbit / Snow Rabbit in Snow Lotus in Tianshan Mountains.

[0059] 3. Identification of adulteration of jellyfish snow rabbit / cotton-headed snow rabbit in Tianshan Snow Lotus

[0060] 1. DNA extraction

[0061] Take the above-mentioned fine powder of the jellyfish snow rabbit, cotton-headed snow rabbit and Tianshan snow lotus samples, accurately weigh according to the proportion in Table 3 below, and mix evenly (a total of 0.1g); take 20mg, put it in a 1.5ml centrifuge tube, and use the plant genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract nucleic acid in the same way to obtain a DNA template.

[0062] Table 3 Mixing ratio of jellyfish snow rabbit, cotton-headed snow rabbit and Tianshan snow lotus

[0063] serial number Jellyfish Snow Rabbit / Snow Rabbit Mass (g) Tianshan Snow Lotus Mass (g) Adulteration ratio 1 0.001 0.099 1％ 2 0.005 0.195 2.5％ 3 0.005 0.095 5％ 4 0.010 0.090 10％ 5 0.025 0.075 25％ 6 0.050 0.050 50％ 7 0.100 / 100％

[0064] (II) Fluorescence PCR amplification reaction

[0065] The DNA template obtained above was added to the PCR reaction system for fluorescent PCR amplification reaction and CT value analysis, wherein the PCR reaction system is shown in Table 4 below.

[0066] Table 4 PCR reaction system

[0067]

[0068] In addition, the PCR amplification reaction program was as follows: 94°C / 3min, 94°C / 5sec, 62.5°C / 30sec, including 94°C / 5sec, 62.5°C / 30sec.

[0069] (III) CT value analysis

[0070] The PCR amplification products were analyzed using a Bio-Rad CFX96 analyzer. The CT values ​​of samples with different adulteration ratios were analyzed, and the relationship between the absolute value of the CT value difference (△CT absolute value) of samples with different adulteration ratios and positive samples was analyzed with 100% adulteration of the mass of the jellyfish snow rabbit / cotton head snow rabbit as the positive control and the adulteration ratio was analyzed.

[0071] Since the polymerase chain reaction is exponentially amplified, the number of amplification rounds when the reading value is reached is the CT value: the difference between the CT values ​​of the standard (S) and the test sample (T) is represented by △CT, and the proportion of the target DNA in the test sample can be expressed as:

[0072] Test sample (T) / standard sample (S) % = 2 -△CT绝对值 ×100%

[0073] According to the above formula, the calculated values ​​of the proportion of quality DNA of jellyfish snow rabbit / cotton-headed snow rabbit under different adulteration ratios were calculated. The calculation results are shown in Tables 5 and 6.

[0074] Table 5 Relationship between the actual adulteration ratio of jellyfish snow rabbit and the absolute value of △CT

[0075] Mixed samples Ct mean △Ct Adulteration ratio (calculated value) % Adulteration ratio (expected value) % 1 28.40 7.09 0.74 1.0 2 26.83 5.51 2.19 2.5 3 26.16 4.85 3.47 5 4 25.14 3.83 7.05 10 5 23.52 2.21 21.66 25 6 22.22 0.90 53.46 50 7 21.31 0.00 100.00 100

[0076] Table 6 The relationship between the actual adulteration ratio of Cotton Head Snow Rabbit and the absolute value of △CT

[0077] Mixed samples Ct mean △Ct Adulteration ratio (calculated value) % Adulteration ratio (expected value) % 1 28.52 6.84 0.88 1.0 2 27.34 5.66 1.98 2.5 3 26.46 4.78 3.63 5 4 25.21 3.53 8.66 10 5 24.09 2.41 18.77 25 6 22.91 1.23 42.63 50 7 21.68 0.00 100.00 100

[0078] In actual testing, considering that medicinal materials are allowed to have impurities within 3%, and taking full account of the errors in testing, when the actual adulteration is 5%, the △CT of the jellyfish snow rabbit is 4.85, and the △CT of the cotton-head snow rabbit is 4.78. When the actual adulteration is 10%, the △CT of the jellyfish snow rabbit is 3.83, and the △CT of the cotton-head snow rabbit is 3.53. Therefore, the absolute value of △CT ≤4 is used as the basis for detection.

[0079] IV. PCR Amplification Accuracy and Sensitivity Test

[0080] (I) Method sensitivity investigation

[0081] We selected a batch of genomic DNA from the jellyfish snow rabbit for investigation and used sterile double distilled water for gradient dilution until the DNA template concentration was 100 ng·μL –1 , 50ng·μL –1 , 20ng·μL –1 , 10ng·μL –1 , 1ng·μL –1 and 0.1 ng·μL –1 1 μL of the above templates of different concentrations were taken for amplification using the above-constructed PCR conditions. The results are as follows: Figure 2 As shown, the results showed that when the jellyfish snow rabbit template DNA concentration was diluted to 0.1ng·μL –1 When the template DNA concentration is lower than 0.1 ng·μL –1 When , the Ct value of the amplification curve is greater than 32. The above shows that the method of the present invention has good sensitivity.

[0082] (II) Method specificity investigation

[0083] The fluorescent quantitative PCR primers and specific probes of the present invention were used to perform amplification reactions on 20 batches of Saussurea involucrata, Snow Rabbit, and Snow Rabbit in Table 1 according to the above method, and specificity was verified. Sterile double distilled water was set as a blank control. The results showed that the DNA of the counterfeit Snow Rabbit and Snow Rabbit showed a typical S-shaped amplification curve, while the authentic Saussurea involucrata and the blank control did not show amplification, indicating that the primers and probes screened in the experiment have strong specificity. Figures 3 to 5 .

[0084] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A PCR primer pair for identifying adulteration of Saussurea involucrata, characterized in that: The nucleotide sequences thereof are shown in SEQ ID NO:1 and SEQ ID NO:

2.

2. A PCR primer pair and probe for identifying adulteration of Saussurea involucrata, characterized in that: The nucleotide sequences of the PCR primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequence of the probe is shown in SEQ ID NO:

3.

3. A detection reagent for identifying adulteration of Saussurea involucrata, characterized in that: The method comprises the PCR primer pair for identifying adulteration of Saussurea involucrata described in claim 1 or the PCR primer pair and probe for identifying adulteration of Saussurea involucrata described in claim 2.

4. A detection kit for identifying adulteration of Saussurea involucrata, characterized in that: The invention comprises the detection reagent for identifying adulteration of Saussurea involucrata as described in claim 3.

5. Use of the PCR primer pair for identification of adulteration of Saussurea involucrata according to claim 1 or the PCR primer pair and probe for identification of adulteration of Saussurea involucrata according to claim 2 in the preparation of a detection product for identification of adulteration of Saussurea involucrata.

6. A method for identifying adulterated snow rabbit jellyfish and / or snow rabbit cotton head in Tianshan Saussurea involucrata, characterized in that: include: (1) extracting DNA from the sample to be tested and the standard sample respectively to obtain a DNA template of the sample to be tested and a DNA template of the standard sample; (2) using the PCR primer pair and probe for adulteration identification of Saussurea involucrata described in claim 2 to perform PCR amplification on the DNA template of the sample to be tested and the DNA template of the standard sample to obtain a first CT value and a second CT value; (3) calculating the difference △CT between the first CT value and the second CT value, and identifying the content of adulterated Saussurea jellyfish and / or Saussurea scaly in Saussurea involucrata based on the absolute value of △CT.

7. The identification method according to claim 6, characterized in that: When the absolute value of △CT is ≤4, the content of adulterated Snow Rabbit Jellyfish and / or Snow Rabbit Cotton Head in Tianshan Snow Lotus is ≥10%.

8. The identification method according to claim 6 or 7, characterized in that: The PCR amplification procedure included: 94°C / 3min, 94℃ / 5sec, 62.5℃ / 30sec, of which 94℃ / 5sec, 62.5℃ / 30sec.

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