Kit for identifying bulbus fritillariae cirrhosae and application thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine identification, in particular to a kit for identifying bulbus fritillariae cirrhosae and application thereof. In the kit provided by the invention, at least one of a primer pair consisting of an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2, a primer pair consisting of an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4 and a primer pair consisting of an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO.6 is adopted. When the primer pair is used for PCR amplification, variation sites in an amplification region are relatively stable, so that the bulbus fritillariae cirrhosae can be effectively distinguished from other common bulbus fritillariae cirrhosae. By combining the primers with a PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) technology or a DNA (Deoxyribose Nucleic Acid) macro bar code technology, the bulbus fritillariae cirrhosae

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine identification, in particular to a kit for identifying Fritillaria cirrhosae and application thereof. Background Art

[0002] Chuanbeimu is the dried bulb of Fritillaria cirrhosa D.Don, Fritillaria unibracteata Hsiao et KCHsia, Fritillaria przewalskiiMaxim., Fritillaria delavayi Franch., Fritillaria taipaiensis P.Y.Li or Fritillaria unibracteata Hsiao et KCHsia var.Waubonsie (SY.Tang et S C.Yue) ZDLiu, S.Wanget SCChen. It has the main effects of clearing heat and moistening the lungs, resolving phlegm and relieving cough. However, the annual output of Chuanbeimu is low and expensive, which leads to the problem of mixed adulteration of Chuanbeimu and multi-species originals with other low-priced close relatives (such as flat Fritillaria, Yi Fritillaria and Zhejiang Fritillaria) in the market. In the Chinese patent medicine containing Chuanbeimu, there is also the problem of adulteration.

[0003] Traditional identification methods such as property identification, microscopic identification and physical and chemical identification mainly use the appearance, microscopic characteristics or chemical composition of traditional Chinese medicine as the basis for identification, which are easily affected by many factors such as growth environment, harvest time, human activities and processing. In order to ensure the quality control and safety of Fritillaria cirrhosa and its Chinese patent medicine, a faster, more accurate, efficient and stable method is urgently needed to identify it.

[0004] PCR-RFLP (PCR-restriction fragment length polymorphisms) uses PCR technology to amplify target DNA fragments, then uses restriction endonucleases to recognize specific DNA sites for cutting, and finally uses gel electrophoresis technology to analyze the digestion products, and by comparing the length polymorphism of the digestion fragments, the difference analysis of gene sequences from different sources is realized. The PCR-RFLP technology recorded in the pharmacopoeia can only distinguish samples into non-enzyme-cutting groups (including flat Fritillaria, Zhejiang Fritillaria and Ili Fritillaria) and enzyme-cutting groups (including thick honey Fritillaria, Chinese Fritillaria, Kangding Fritillaria, long gland Fritillaria and Sichuan Fritillaria) according to the variation of a single enzyme-cutting site, and cannot be further distinguished. And in reality, impurities in the sample (such as excipients in Chinese patent medicines) interfere with the enzymatic reaction, which will cause the DNA to be unable to be sheared and detected by electrophoresis after the enzyme-cutting reaction is thoroughly carried out, that is, false negatives that are misjudged as adulteration occur. When the sample DNA is severely degraded, it will also be difficult to detect Sichuan Fritillaria using the PCR-RFLP method. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a kit for identifying Fritillaria cirrhosa and its application. The kit provided by the present invention has high specificity and can quickly and accurately identify Fritillaria cirrhosa and its counterfeit products.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a kit for identifying Fritillaria cirrhosa, comprising at least one primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2, a primer pair consisting of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4, and a primer pair consisting of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6.

[0008] The sequence of SEQ ID NO. 1 is: 5′-ATTCGAATGTATCAAC-3′;

[0009] The sequence of SEQ ID NO. 2 is: 5′-GAGATAATTTGTCCTC-3′;

[0010] The sequence of SEQ ID NO.3 is: 5′-GCTGGTGCTCCGTCCAC-3′;

[0011] The sequence of SEQ ID NO.4 is: 5′-TTGCGAGAYGRCACGCCCT-3′.

[0012] The sequence of SEQ ID NO.5 is: 5′-CGGATGACACCGTGTC-3′;

[0013] The sequence of SEQ ID NO.6 is: 5′-GTCATATGTTCCTTGG-3′.

[0014] Through amplification and sequencing of standard material powders of Fritillaria cirrhosa, Fritillaria foetida, Fritillaria sibiricum and Fritillaria thunbergii, it was verified that the mutation sites in the amplification region of the above primer pairs are relatively stable and can effectively distinguish Fritillaria cirrhosa from other common Fritillaria.

[0015] The primer pair consisting of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2 is artificially designed based on the study of the matK genome of the Fritillaria chloroplast. Through the amplification and sequencing verification of the standard material powder of Fritillaria cirrhosa, Fritillaria dahurica, Fritillaria foetida and Fritillaria thunbergii, the above primer pair can not only distinguish Fritillaria cirrhosa from other common Fritillaria, but also simultaneously identify Fritillaria cirrhosa, Fritillaria dahurica, Fritillaria thunbergii and Fritillaria thunbergii from mixed and counterfeit products. It is applicable even in the case of great differences in the proportion of species, and has good quantitative ability. It can be used not only for the identification of Fritillaria cirrhosa medicinal materials and their counterfeits and mixed counterfeits, but also for the identification of Fritillaria cirrhosa and its counterfeits in Chinese patent medicines.

[0016] The above primer pair consisting of the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4 is artificially designed based on the ITS2 region DNA sequence of the Fritillaria species. It can distinguish the Fritillaria cirrhosa medicinal materials and their counterfeits through DNA macrobarcoding technology, and can distinguish the Fritillaria cirrhosa and its counterfeits in Chinese patent medicines.

[0017] The above primer pair consisting of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2 and the primer pair consisting of the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4 have species resolution comparable to that of the full-length ITS2 and have higher amplification efficiency for Fritillaria cirrhosae than traditional barcodes.

[0018] The primer pair consisting of the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6 is artificially designed based on the DNA sequence of the ITS region of the Fritillaria species. It can distinguish Fritillaria cirrhosa and its counterfeits by PCR-RFLP method, and can also distinguish Fritillaria cirrhosa medicinal materials and its counterfeits by DNA macrobarcoding technology. Compared with the primers in the pharmacopoeia, the amplification ability of this primer pair is stronger. By using this primer pair for PCR amplification, combined with SmaI restriction enzyme digestion and Sanger sequencing technology, the identification of Fritillaria cirrhosa medicinal materials can be accurately achieved.

[0019] In combination with the first aspect, the kit includes a primer pair consisting of an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2. The primer pair has a stronger resolution ability and can not only determine whether there are counterfeits mixed in the test substance, but also determine the specific type of the counterfeits.

[0020] In combination with the first aspect, the kit includes a primer pair consisting of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4.

[0021] In combination with the first aspect, the kit further comprises a PCR buffer, a DNA polymerase and a template DNA, wherein the PCR buffer contains magnesium ions and deoxynucleoside triphosphates (dNTPs).

[0022] In combination with the first aspect, the kit includes a primer pair consisting of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6.

[0023] Preferably, the kit further comprises restriction endonuclease Sma I.

[0024] Preferably, the kit further comprises a PCR buffer, a DNA polymerase, a template DNA and a restriction endonuclease Sma I; the PCR buffer contains magnesium ions and deoxynucleoside triphosphates.

[0025] The above PCR buffer, DNA polymerase and enzyme digestion buffer can all be selected from commercially available reagents for PCR amplification and enzyme digestion. For example, Gflex from Bio-Rad Biotechnology Co., Ltd. can be selected. TM Buffer, Tks Gflex TM DNA Polymerase and 10×T Buffer. Template DNA is the total DNA of the sample to be tested.

[0026] In a second aspect, the present invention also provides the use of the above-mentioned kit in identifying Fritillaria cirrhosa and its counterfeits.

[0027] In conjunction with the second aspect, the specific steps of the application include:

[0028] S1. Extracting total DNA of the sample to be tested;

[0029] S2. Using the total DNA as template DNA, perform PCR amplification with the above primers, and identify Fritillaria cirrhosa and its counterfeits based on the amplification results.

[0030] Preferably, the reaction system of the PCR amplification is:

[0031]

[0032] For the primer pair consisting of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2, the amplification reaction conditions were: 98°C, 1 min; 98°C, 10 s, 48°C, 15 s, 68°C, 30 s, 40 cycles; 68°C, 5 min;

[0033] For the primer pair consisting of the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4, the amplification reaction conditions were: 98°C, 1 min; 98°C, 10 s, 62°C, 15 s, 68°C, 30 s, 40 cycles; 68°C, 5 min;

[0034] For the primer pair consisting of the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6, the amplification reaction conditions are: 98°C, 1 min; 98°C, 10 s, 45°C, 15 s, 68°C, 30 s, 40 cycles; 68°C, 5 min.

[0035] In the PCR amplification reaction system, the concentration of the template DNA can be routinely adjusted according to the effect of PCR amplification.

[0036] In combination with the second aspect, the application is for identifying Fritillaria cirrhosa and its counterfeits in Chinese patent medicines, and the kit includes at least one primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2 and a primer pair consisting of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4.

[0037] In conjunction with the second aspect, the application includes identifying specific types of counterfeits, and the kit includes a primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2.

[0038] Preferably, the specific step of the application further comprises digesting the PCR amplification product with restriction endonuclease Sma I, and identifying by the digestion result: the sample to be tested whose PCR amplification product can be digested is Fritillaria cirrhosa, otherwise it is a counterfeit.

[0039] Preferably, the reaction system of the enzyme digestion is:

[0040]

[0041] The enzyme digestion reaction conditions were: 30°C water bath for two hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1The NJ tree of the DNA mini-barcode region of Fritillaria cirrhosa, Fritillaria thunbergii, Fritillaria irritans and Fritillaria pacifica in Example 11 of the present invention;

[0043] Figure 2 Constructing an NJ tree for the Fritillaria standard substance sequence and the reference sequence in Example 11 of the present invention;

[0044] Figure 3 This is a correlation analysis between the mixed ratio of Fritillaria cirrhosa and Fritillaria cirrhosa and the proportion of the number of sequences in Example 12 of the present invention;

[0045] Figure 4 This is a correlation analysis between the mixed sample ratio of Fritillaria cirrhosa and Fritillaria cirrhosa and the proportion of sequence numbers in Example 12 of the present invention;

[0046] Figure 5 This is a correlation analysis between the mixed sample ratio of Fritillaria thunbergii and Fritillaria cirrhosa and the proportion of sequence numbers in Example 12 of the present invention;

[0047] Figure 6 This is a correlation analysis of the proportion of mixed samples of Fritillaria cirrhosa, Fritillaria foetida, Fritillaria thunbergii and Fritillaria cirrhosa and the proportion of the number of sequences in Example 12 of the present invention;

[0048] Figure 7 This is a correlation analysis of the proportion of the mixed samples of Fritillaria cirrhosa and other Fritillaria cirrhosa and the proportion of the number of sequences in Example 12 of the present invention;

[0049] Figure 8 This is the amplification result of the commercially available Fritillaria cirrhosa medicinal material in Example 13 of the present invention;

[0050] Fig. 9 The NJ tree of the commercially available Fritillaria cirrhosae medicinal material sequence and the reference sequence in Example 13 of the present invention;

[0051] Fig.10 This is the NJ tree of the OTU sequence and the Fritillaria reference sequence in Example 14 of the present invention;

[0052] Fig.11 The NJ tree (part) of the full-length ITS2 in Example 15 of the present invention and the primer pair in Example 2;

[0053] Fig.12 The agarose gel electrophoresis result in Example 16 of the present invention;

[0054] Fig.13 is the dilution curve in Example 16 of the present invention;

[0055] Fig.14 The PCR amplification results of the primers in Example 17 of the present invention on the Fritillaria cirrhosa medicinal material;

[0056] Fig.15 The enzymatic digestion result of the commercially available Fritillaria cirrhosa medicinal material in Example 17 of the present invention;

[0057] Fig.16 The result of enzymatic digestion of the Chinese patent medicine containing Fritillaria cirrhosa in Example 17 of the present invention;

[0058] Fig.17 This is the Sanger sequencing result of the commercially available Fritillaria cirrhosa medicinal material using the primers in Example 17 of the present invention. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0060] Fritillaria cirrhosa has the main effects of clearing heat and moistening the lungs, resolving phlegm and relieving cough, and is one of the commonly used Chinese medicines. However, the annual production of Fritillaria cirrhosa is low and the price is expensive, which leads to adulteration problems in Fritillaria cirrhosa medicinal materials and Chinese patent medicines containing Fritillaria cirrhosa. Methods based on sequencing technology provide a more objective basis for identification. Super barcodes such as complete chloroplast genomes can distinguish different Fritillaria species, but the overly long fragments make it impossible to obtain the barcode region through PCR. Each decoction sample must be separately library-built and sequenced, with high experimental costs and low throughput. The DNA barcode method based on first-generation sequencing is completely unsuitable for the detection of mixed samples, and the amplicon length is generally greater than 500bp, which also limits the application of universal DNA barcode molecular markers in samples with severe DNA degradation.

[0061] In order to achieve the identification of Fritillaria cirrhosa in Sichuan medicinal materials and Chinese patent medicines, the present invention provides a kit for identifying Fritillaria cirrhosa and its application. The kit includes at least one primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2, a primer pair consisting of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4, and a primer pair consisting of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6. The target fragments of the above primer pairs are all short in length, and no separate library construction and sequencing are required. High throughput can be used to achieve identification, greatly improving the efficiency of identification.

[0062] Pharmacopoeia uses PCR-RFLP to identify it, but this method can only distinguish Fritillaria cirrhosa from other Fritillaria cirrhosa based on enzyme cutting sites, but it is difficult to further determine whether the other Fritillaria cirrhosa is specifically Fritillaria pingensis, Fritillaria thunbergii, or Fritillaria yi, and it is difficult to apply to mixed samples and samples with severe DNA degradation (such as adulterated Fritillaria cirrhosa powder and Fritillaria cirrhosa Chinese patent medicine products), so it is difficult to apply to the identification of Fritillaria cirrhosa in Chinese patent medicines. Known universal DNA barcodes such as ITS, ITS2, psbA-trnH, matK, etc. can distinguish genuine Fritillaria cirrhosa from mixed and counterfeit products, but cannot distinguish genuine Fritillaria cirrhosa to the species level.

[0063] In the kit provided by the present invention, the primer pair consisting of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2, and the primer pair consisting of the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4 can both be applied to the identification of Fritillaria cirrhosa in mixed samples and samples with severe DNA degradation, wherein the primer pair consisting of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2 can not only distinguish Fritillaria cirrhosa from other common Fritillaria cirrhosa, but also identify the specific species of counterfeit Fritillaria cirrhosa.

[0064] In the kit provided by the present invention, the primer pair consisting of the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6 can be used to distinguish Fritillaria cirrhosa and its counterfeit by PCR-RFLP method, and can also be used to distinguish Fritillaria cirrhosa and its counterfeit by DNA macrobarcoding technology, which is convenient for selection in combination with experimental conditions in practical applications. When the primer pair in the kit is this primer pair, it can also include restriction endonuclease Sma I. The PCR amplification primer of Fritillaria cirrhosa can be digested by enzyme, while the PCR amplification primer of non-Fritillaria cirrhosa cannot be digested by enzyme, and the digestion result can be used to distinguish Fritillaria cirrhosa and non-Fritillaria cirrhosa.

[0065] The following is further described with reference to specific embodiments.

[0066] The Fritillaria cirrhosa and other common Fritillaria standard substance powders used in the following examples were purchased from the National Standard Material Platform. Specific information is shown in Table 1.

[0067] Table 1 Information of standard substances of Fritillaria cirrhosa

[0068]

[0069] The specific information of the Fritillaria cirrhosa medicinal materials and the Chinese patent medicines containing Fritillaria cirrhosa used in the following examples are shown in Tables 2 and 3.

[0070] Table 2 Information of Fritillaria cirrhosa medicinal material samples

[0071]

[0072]

[0073] Table 3 Information of Chinese patent medicine samples containing Fritillaria cirrhosa

[0074]

[0075]

[0076] The main instruments used in the following examples are shown in Table 4

[0077] Table 4 Main instruments of the experiment

[0078]

[0079]

[0080] The main experimental reagents used in the following examples are shown in Table 5.

[0081] Table 5 Main experimental reagents

[0082]

[0083]

[0084] Example 1

[0085] The embodiment of the present invention provides a kit for identifying Fritillaria cirrhosae, comprising a primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2.

[0086] Example 2

[0087] The embodiment of the present invention provides a kit for identifying Fritillaria cirrhosae, comprising a primer pair consisting of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4.

[0088] Example 3

[0089] The embodiment of the present invention provides a kit for identifying Fritillaria cirrhosae, comprising a primer pair consisting of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6.

[0090] Example 4

[0091] The embodiment of the present invention provides a kit for identifying Fritillaria cirrhosa, comprising a primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2, and a primer pair consisting of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4.

[0092] Example 5

[0093] The embodiment of the present invention provides a kit for identifying Fritillaria cirrhosa, comprising a primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2 and a primer pair consisting of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6.

[0094] Example 6

[0095] The embodiment of the present invention provides a kit for identifying Fritillaria cirrhosa, comprising a primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2, an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4, and a primer pair consisting of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6.

[0096] Example 7

[0097] The embodiment of the present invention provides a kit for identifying Fritillaria cirrhosa, comprising:

[0098] (1) A primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2.

[0099] (2) PCR reaction reagent: Gflex TM Buffer, Tks Gflex TM DNA Polymerase and template DNA.

[0100] Example 8

[0101] The embodiment of the present invention provides a kit for identifying Fritillaria cirrhosa, comprising:

[0102] (1) A primer pair consisting of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4.

[0103] (2) PCR reaction reagent: Gflex TM Buffer, Tks Gflex TMDNA Polymerase and template DNA.

[0104] Example 9

[0105] The embodiment of the present invention provides a kit for identifying Fritillaria cirrhosa, comprising:

[0106] (1) A primer pair consisting of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.6.

[0107] (2) PCR reaction reagent: Gflex TM Buffer, Tks Gflex TM DNA Polymerase and template DNA.

[0108] Example 10

[0109] The embodiment of the present invention provides a kit for identifying Fritillaria cirrhosa, comprising:

[0110] (1) A primer pair consisting of an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2, and restriction endonuclease Sma I.

[0111] (2) PCR reaction reagent: Gflex TM Buffer, Tks Gflex TM DNA Polymerase and template DNA.

[0112] (3) Enzyme cleavage reaction reagent: T Buffer.

[0113] Embodiment 11

[0114] This example provides a method for identifying Fritillaria cirrhosa, and the primer pair in Example 1 is used to identify Fritillaria cirrhosa.

[0115] 1. DNA extraction and detection

[0116] 1.1 DNA extraction

[0117] DNA extraction was performed using TIANGEN's plant genomic DNA extraction kit. The specific operation process is as follows:

[0118] (1) Weigh approximately 100 mg of each sample, grind thoroughly, and place in a centrifuge tube.

[0119] (2) Add 700 μl of buffer GP1 (add 0.1% mercaptoethanol to GP1 preheated at 65°C before the experiment), invert to mix, and place the centrifuge tube in a 65°C water bath for 20 min. Invert the centrifuge tube several times during the water bath to mix;

[0120] (3) Add 700 μl of chloroform, mix thoroughly, and centrifuge at 12,000 rpm (Revolutions per minute) for 5 min;

[0121] (4) Transfer the upper aqueous phase obtained in the previous step to a new centrifuge tube, add 700 μl of buffer GP2, and mix thoroughly;

[0122] (5) Transfer the mixed liquid to the adsorption column, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid;

[0123] (6) Add 500 μl of buffer GD to the adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and then return the adsorption column to the collection tube;

[0124] (7) Add 600 μl of rinse solution PW to the adsorption column, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column in a collection tube;

[0125] (8) Repeat step (7);

[0126] (9) Place the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 2 min, discard the waste liquid in the collection tube, and place the adsorption column at room temperature for 10 min to remove the residual ethanol in the adsorption material;

[0127] (10) Transfer the adsorption column to a clean centrifuge tube, add 60 μl of elution buffer TE in the middle of the adsorption membrane, let it stand at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, and then transfer the solution to a new centrifuge tube to obtain the DNA of each sample.

[0128] 1.2 DNA extraction results detection

[0129] The concentration and purity of the extracted total DNA were determined using a NanoDrop 2000 instrument, and the degradation of the sample DNA was observed by agarose gel electrophoresis.

[0130] 2. Primer information

[0131] The DNA mini-barcode primers used in this example are the primer pair provided in Example 1. Detailed information of the primers is shown in Table 6.

[0132] Table 6 Primer information (SEQ ID NO.1, SEQ ID NO.2)

[0133]

[0134] According to the conventional operation of primer dilution, the above primers were diluted to 10 μM with ddH2O.

[0135] MEGA was used to construct an NJ tree for the DNA mini-barcode region. The NJ tree results showed that the DNA mini-barcode region could cluster Fritillaria thunbergii, Fritillaria thunbergii, and Fritillaria cirrhosa into one branch, and Fritillaria cirrhosa and other Fritillaria could be distinguished. Figure 1 , indicating that the primers designed in Example 1 of the present invention meet the requirements.

[0136] 3. PCR amplification and sequencing

[0137] The PCR reaction system is shown in Table 7, and the amplification program is shown in Table 8.

[0138] Table 7 PCR reaction system

[0139]

[0140] Note: The total reaction volume is 50 μL.

[0141] Table 8 PCR amplification program

[0142]

[0143] A negative control was added to each PCR experiment. After the PCR reaction was completed, the PCR product was observed by 1.5% agarose gel electrophoresis. The success of PCR was detected by agarose gel electrophoresis, and the electrophoresis voltage was set to 110V and the electrophoresis time was set to 30min.

[0144] 4. Sanger sequencing and species identification

[0145] The PCR products of each sample were sequenced using the Sanger method, and the sequencing data were spliced ​​using Geneious Prime 2020.2. The consensus sequence after splicing was compared with the reference sequence, and the sequencing results of each Fritillaria standard substance were compared with the reference sequence to build an NJ tree. The results showed that the sequencing result sequence of the Fritillaria cirrhosa standard substance was 100% consistent with the Fritillaria cirrhosa reference sequence, and the plain Fritillaria, Ilex iruri, and Fritillaria thunbergii could also be completely aligned with their reference sequences, and they were all clustered into one branch with their reference sequences on the NJ tree (see the results). Figure 2 ), indicating that the mutation site is relatively stable and the primers can well distinguish Fritillaria cirrhosa from other Fritillaria.

[0146] Example 12

[0147] This example investigates the minimum detection limit of the primer pair of Example 1 for identifying Fritillaria cirrhosa.

[0148] 1. Materials

[0149] The Fritillaria cirrhosa standard substance powder was mixed with other Fritillaria cirrhosa standard substance powders. The specific information is shown in Table 9.

[0150] Table 9 Mixed sample information of Fritillaria cirrhosa reference material

[0151]

[0152]

[0153] 2. DNA extraction and detection: Same as Example 11

[0154] 3. Primer information:

[0155] Same as Example 11. During amplification, different tag sequences of 8 bp are added to the 5′ ends of the upstream and downstream primers to distinguish each sample during data analysis.

[0156] According to the conventional operation of primer dilution, the above primers were diluted to 10 μM with ddH2O.

[0157] 4. PCR amplification and sequencing:

[0158] The PCR reaction system, amplification procedure, and electrophoresis method were the same as those in Example 11.

[0159] All PCR products were mixed in equimolar amounts and used the kit Ultra TM DNA LibraryPrep Kit for Illumina (NEB, USA) was used to generate a sequencing library. Sequencing was performed on the Illumina NovaSeq platform with a read length of 2 × 150 bp.

[0160] 5. Amplicon data processing

[0161] 5.1 Data quality control and double-end splicing

[0162] The sequencing data was removed from the machine and the sequencing adapters were preliminarily controlled to generate clean data. For the obtained clean data, FastQC (www.bioinformatics.babraham.ac.uk / projects / fastqc / ) was first used to perform quality detection on the paired-end data to observe whether the base quality, length distribution, and removal of adapter contamination of the clean data were qualified. After confirming that the data was qualified, Trimmomatic was used to further screen the clean data for quality, so that the average base quality was not less than 20. The join_paired_ends.py script was used to merge the paired-end data.

[0163] 5.2 Data Splitting and Primer Removal

[0164] Use the extract_barcodes.py script to extract the barcode, then use the split_libraries_fastq.py script to split the sample data, and then use the cutadapt command to remove the primer sequence.

[0165] 5.3 Sequence removal of phix pollution, redundancy and noise

[0166] The filter_phix command of USEARCH was used to remove phix contamination from the sequences. The fastx_uniques algorithm and the option “minuniquesize 12” were used to remove redundancy so that only one read with exactly the same base sequence was retained. The USEARCH UNOISE3 algorithm was applied to detect and remove chimeras, and the awk command was used to modify the sequence name format.

[0167] 5.4 Generate OTU table and representative sequences

[0168] Use USEARCH to cluster OTUs with 100% similarity to generate OTU representative sequences, and use the data after removing phix contamination to map OTU representative sequences to generate an OTU table. Then use QIIME to delete OTUs with a relative abundance of less than 0.01% from the OTU table and regenerate the OTU table and OTU representative sequences.

[0169] 5.5 Sequence Alignment

[0170] The obtained OTU result sequence was imported into Geneious Prime 2020.2 software for comparison and analysis with the reference sequence.

[0171] 6. Experimental results

[0172] 6.1 Sequencing data analysis

[0173] All mixed samples were amplified successfully and sequenced. After analysis, a total of 4 OTUs were obtained (OTUs were clustered using USEARCH with 100% similarity) and 1,220,042 sequences. The alignment results showed that OTU_1 was Fritillaria cirrhosa, OTU_2 was Fritillaria dasyphylla, OTU_3 was Fritillaria irritans, and OTU_4 was Fritillaria thunbergii. The sequence numbers of the sequencing results are shown in Table 10.

[0174] Table 10 Sequence number of each sample

[0175]

[0176] 6.2 Qualitative and quantitative capability analysis

[0177] In all mixed samples, Fritillaria cirrhosa, Fritillaria foetida, Fritillaria ilium and Fritillaria thunbergii can be identified. It is applicable even when the species proportions vary greatly (such as samples 5, 10 and 15, in which the biomass of Fritillaria cirrhosa, Fritillaria ilium and Fritillaria thunbergii is about 3.3%). On the one hand, this shows that within a larger threshold range (0.033-0.933), the primer pair of Example 1 of the present invention has good qualitative identification ability. On the other hand, it also shows that the primer pair of Example 1 has a high sensitivity and can achieve effective detection of trace components in mixed samples.

[0178] To further verify the ability of the primer pair in Example 1 to identify Fritillaria cirrhosa, this example conducted a correlation analysis between the sequence number ratio and biomass ratio in the experiment in which Fritillaria cirrhosa was mixed with Fritillaria cirrhosa, Fritillaria foetida, and Fritillaria thunbergii in a certain proportion, and Fritillaria cirrhosa and these three common Fritillaria in a certain proportion. Figure 3 to Figure 7 The results showed that the proportion of the sequence numbers of Fritillaria cirrhosa, Fritillaria cirrhosa, and Fritillaria foetida was linearly correlated with biomass, indicating that the primer pair can perform good quantitative analysis on the three Fritillaria species in the mixed sample. When there was only the Fritillaria thunbergii sample, the number of reads obtained by sequencing was not small, but when the Fritillaria thunbergii was mixed with other Fritillaria, the number of reads obtained by sequencing was relatively small, which suggests that the primer pair may have amplification preference for Fritillaria thunbergii.

[0179] Embodiment 13

[0180] This example investigates the application of the primer pair of Example 1 in identifying Fritillaria cirrhosa.

[0181] 1. PCR amplification

[0182] According to the method of Example 11, PCR amplification was performed on commercially available Fritillaria cirrhosae (shown in Table 2). The sample amplification results are shown in Figure 8 (In the figure, 1 to 15 are PCR products of 15 commercially available Fritillaria cirrhosae medicinal materials; K is an empty tube). The gel electrophoresis results showed that all 15 commercially available Fritillaria cirrhosae medicinal materials were successfully amplified.

[0183] 2. Sanger sequencing results

[0184] The sequencing results were spliced ​​with double-end De Novo Assemble and compared with the reference sequence. The sequencing results of each Fritillaria cirrhosa sample were compared with the reference sequence to build an NJ tree. The results are shown in Fig. 9Samples 1 to 9 were 100% consistent with the reference sequence of Fritillaria cirrhosae, and were clustered into one branch with their reference sequence on the NJ tree, and were identified as Fritillaria cirrhosae. Samples 10 to 14 were consistent with the reference sequence of Fritillaria cirrhosae, and were clustered into one branch with their reference sequence on the NJ tree, and were identified as Fritillaria cirrhosae. Sample 15 was consistent with the reference sequence of Fritillaria thunbergii, and was clustered into one branch with its reference sequence on the NJ tree, and was identified as Fritillaria thunbergii.

[0185] Embodiment 14

[0186] This example investigates the application of the primer pair of Example 1 in identifying Fritillaria cirrhosa in Chinese patent medicine.

[0187] The commercially available Chinese patent medicine (shown in Table 3) was PCR amplified and sequenced according to the method of Example 12, and the amplicon data was processed. When amplifying the Chinese patent medicine, different tag sequences of 8 bp were added to the 5' ends of the upstream and downstream primers to distinguish each sample during data analysis.

[0188] Experimental results:

[0189] All 28 samples were successfully amplified and sequenced. After analysis, a total of 10 OTUs (OTUs were clustered using USEARCH with 100% similarity) and 1,111,139 sequences were obtained. The rarefaction curve tended to be flat and stable, and the number of OTUs in each sample gradually stabilized. This shows that the sequencing depth is sufficient to reveal the species diversity of the samples.

[0190] The obtained 10 OTUs were used to construct the NJ tree with the Fritillaria reference sequence. The results are shown in Fig.10 The comparison results showed that OTU_2, OTU_3 and OTU_10 were clustered with Fritillaria cirrhosa and identified as Fritillaria cirrhosa; OTU_4 was clustered with Fritillaria thunbergii and identified as Fritillaria thunbergii; OTU_6 and OTU_7 were clustered with Fritillaria ilii and identified as Fritillaria yili; OTU_8 was clustered with Fritillaria dasyphylla and identified as Fritillaria dasyphylla; OTU_1, OTU_5 and OTU_9 could not be determined from the NJ tree results. Blast comparison of these three OTUs in the NCBI database showed that none of these three OTUs belonged to the genus Fritillaria. The OTU comparison and identification results and sequence numbers are shown in Table 11.

[0191] Table 11 OTU alignment and identification results and sequence numbers

[0192]

[0193]

[0194] Embodiment 15

[0195] This example provides a method for identifying Fritillaria cirrhosa, and the primer pair in Example 2 is used to identify Fritillaria cirrhosa.

[0196] 1. DNA extraction and detection: same as Example 11.

[0197] The DNA mini-barcode primers used in this example are the primer pair provided in Example 2. Detailed information of the primers is shown in Table 12.

[0198] Table 12 Primer information (SEQ ID NO.3, SEQ ID NO.4)

[0199]

[0200] During amplification, different tag sequences can be added to the 5' ends of the upstream and downstream primers to distinguish each sample during data analysis.

[0201] According to the conventional operation of primer dilution, the above primers were diluted to 10 μM with ddH2O.

[0202] MEGA was used to construct an NJ tree for the DNA mini-barcode region. The cluster analysis results showed that the six authentic Fritillaria species and other Fritillaria species were clustered on different branches, which can effectively distinguish Fritillaria from its counterfeits. The NJ tree also showed that the species resolution of the above primer pair was equivalent to that of the full-length ITS2, indicating that the primer pair of Example 2 of the present invention is suitable for the identification of severely degraded traditional Chinese medicines containing Fritillaria. The NJ tree of the full-length ITS2 and the primer pair of Example 2 of the present invention (partial) is shown in Fig.11 (The grey sequence in the figure is the authentic Fritillaria cirrhosa sequence, and the black sequence is the counterfeit Fritillaria cirrhosa sequence).

[0203] 2. PCR amplification

[0204] The PCR reaction system is shown in Table 13, and the amplification program is shown in Table 14.

[0205] Table 13 PCR reaction system

[0206]

[0207]

[0208] Note: The total reaction volume is 50 μL.

[0209] Table 14 Amplification program

[0210]

[0211] All samples were repeated three times at the PCR level, A, B, and C, respectively, and negative controls were added to each PCR experiment. After the PCR reaction was completed, the PCR products were observed by 1% agarose gel electrophoresis. Agarose gel electrophoresis was used to detect whether the PCR was successful, and the electrophoresis voltage was set to 110V and the electrophoresis time was 30min.

[0212] Example 16

[0213] This example investigates the application of the primer pair of Example 2 in identifying Fritillaria cirrhosa in Chinese patent medicine.

[0214] 1. PCR amplification and sequencing

[0215] According to the method of Example 15, PCR amplification and sequencing were performed on the commercially available Chinese patent medicine (shown in Table 3). Different tag sequences of 8 bp were added to the 5′ end of the upstream and downstream primers to distinguish each sample during data analysis. The PCR products were excised and recovered to remove non-specific PCR products and primer dimers. Subsequently, the PCR products were mixed in equimolar amounts. The kit was used Ultra TM DNA Library Prep Kit for Illumina (NEB, USA) was used to generate 6 sequencing libraries. Sequencing was performed on the Illumina NovaSeq platform with a read length of 2×150 bp.

[0216] All samples were successfully amplified and sequenced. The agarose gel electrophoresis results of each library are shown in Fig.12 (In the figure, M-2, M-1: DNA marker; 1-6: library 1, library 2, library 3, library 4, library 5, library 6; S: ​​standard loading amount).

[0217] 2. Amplicon data processing

[0218] 2.1 Data splitting and primer removal

[0219] The sequencing company removes the sequencing adapters and performs preliminary quality control on the data from the sequencing machine to generate clean data. For the obtained clean data, FastQC (www.bioinformatics.babraham.ac.uk / projects / fastqc / ) is first used to perform quality detection on the double-end data to observe whether the base quality, length distribution, and removal of adapter contamination of the clean data are qualified. After confirming that the data is qualified, Trimmomatic is used to further screen the quality of the clean data so that the average base quality is not less than 20. Fastq-multx is used to split the sequencing data samples according to different tag sequences; and bbduk from BBMap software is used to remove primers and tag sequences.

[0220] 2.2 Sequence double-end splicing

[0221] The split data were joined using the join_paired_ends.py command of QIIME. The sequence names and sequence formats were converted using the obiconvert script, sed command, seqkit software, and fasta_formatter script.

[0222] 2.3 Sequence length screening

[0223] Sequences with a length of 127 bp were retained using the filter_fasta_by_length script.

[0224] 2.4 Sequence removal of phix pollution, redundancy and noise: same as Example 11.

[0225] 2.5 Generate OTU table and representative sequences: Same as Example 11.

[0226] In this example, after processing the data of 6 libraries, a total of 11 OTUs (OTUs were clustered using USEARCH with 100% similarity) and 12,764,817 sequences were obtained. The number of sequences contained in each sample is shown in Table 15.

[0227] Table 15 Sequence number of each sample

[0228]

[0229]

[0230] 3. Species identification

[0231] BLASTN was used to compare the representative sequences of OTUs with the GenBank database of NCBI (National center for biotechnology information), and the parameters were set as follows: -outfmt5 -evalue 10 -max_target_seqs 10 -gapopen 5 -gapextend 2 -num_threads 16. The alignment results were imported into MEGAN COMMUNITY EDITION 6.10.8 to export taxonomic information with a similarity threshold of 100%, and the parameters were set as follows: Min Score: 50.0, Max Expected: 0.01, Min Percent Identity: 100.0, Top Percent: 10.0, Min Support Percent: 0.01, Min Support: 1, LCA Algorithm: weighted.

[0232] The 11 OTUs obtained were divided into two groups according to the identification results: authentic Fritillaria cirrhosa (group 1) and counterfeit products (group 2). Finally, 6 authentic Fritillaria cirrhosa OTUs with a total of 9,156,453 sequences and 5 counterfeit products OTUs with a total of 3,608,364 sequences were identified. The species identification results are detailed in Table 16.

[0233] Table 16 Species identification results

[0234]

[0235]

[0236] 4. Dilution curve

[0237] The dilution curve was used to evaluate whether the existing sequencing amount was reasonable. Each sample was averaged according to the sequence number of the minimum sample group (BM21-B) of 1,217, and the dilution curve was drawn. The results are shown in Fig.13 As the number of sequence samples increased, the rarefaction curve became flat and stable before the sequencing amount reached 200, and the number of OTUs in each sample gradually stabilized, indicating that the sequencing depth was sufficient to reveal the species diversity of the samples.

[0238] 5. Repeat the test

[0239] The analysis results of the proportion of Fritillaria cirrhosa and its mixed products in various Chinese patent medicine samples showed that the abundance proportions of authentic and fake products were relatively similar in the three repeated experiments, indicating that the results obtained in this experiment are relatively reliable and the experiment is repeatable.

[0240] Embodiment 17

[0241] This example provides a method for identifying Fritillaria cirrhosa, and the primer pair in Example 3 is used to identify Fritillaria cirrhosa.

[0242] 1. DNA extraction and detection: Same as Example 11

[0243] 2. Primer information

[0244] The specific primers used in this example are the primer pairs provided in Example 3. Detailed information of the primers is shown in Table 17.

[0245] Table 17 Primer information

[0246]

[0247] According to the conventional operation of primer dilution, the above primers were diluted to 10 μM with ddH2O.

[0248] 3. PCR amplification and enzyme digestion

[0249] The PCR reaction system is shown in Table 18, and the amplification program is shown in Table 19. The enzyme digestion program is shown in Table 20.

[0250] Table 18 PCR reaction system

[0251]

[0252] Note: The total reaction volume is 50 μL.

[0253] Table 19 PCR amplification program

[0254]

[0255] Table 20 Sma I restriction enzyme system

[0256]

[0257] Note: The total reaction volume is 20 μL.

[0258] A negative control was added to each PCR experiment. After the PCR reaction was completed, the PCR product was observed by 1.5% agarose gel electrophoresis. Agarose gel electrophoresis was used to detect whether the PCR was successful. The electrophoresis voltage was set to 110V and the electrophoresis time was set to 30 minutes. Subsequently, the PCR product was subjected to an enzyme digestion experiment. After the enzyme digestion system was configured, it was placed in a 30°C water bath for two hours. After the enzyme digestion reaction was completed, the enzyme digestion product was observed by 1.5% agarose gel electrophoresis to obtain the enzyme digestion result.

[0259] PCR amplification results are shown in Fig.14(In the figure, 1 to 15 are PCR products of 15 commercially available Fritillaria cirrhosa medicinal materials; F is a negative tube, i.e., a tube with a negative control added). The gel electrophoresis results of the PCR products showed that all 15 commercially available Fritillaria cirrhosa medicinal materials and 28 batches of traditional Chinese medicines containing Fritillaria cirrhosa were successfully amplified.

[0260] The results of enzyme digestion of commercially available Fritillaria cirrhosae are shown in Fig.15 (In the figure, Ping, Chuan, Yi, and Zhe are the standard controls for Ping Fritillaria, Chuan Fritillaria, Yi Fritillaria, and Zhejiang Fritillaria, respectively; 1 to 15 are the digestion products of PCR products of 15 commercially available Fritillaria cirrhosa medicinal materials; F is a negative tube). The digestion results show that Fritillaria cirrhosa will be digested into a band of about 80bp, while non-Fritillaria cirrhosa will not be digested. Among the 15 commercially available Fritillaria cirrhosa medicinal materials samples, 4 samples were digested into short bands and identified as Fritillaria cirrhosa, and 11 samples were still one band and identified as other Fritillaria. The digestion results of traditional Chinese medicine containing Fritillaria cirrhosa are shown in Fig.16 The enzyme digestion results of some Chinese patent medicine samples showed dark or no bands, indicating that the PCR-RFLP method was not suitable for the identification of Fritillaria cirrhosa in Chinese patent medicine samples.

[0261] 4. Sanger sequencing and species identification

[0262] The PCR products of each medicinal material sample were sequenced using the Sanger method, and the sequencing data were spliced ​​using Geneious Prime2020.2 for double-end data De Novo Assembly. The spliced ​​consensus sequence was aligned with the reference sequence for key sites and the entire sequence.

[0263] The restriction site sequence of the authentic Fritillaria cirrhosa is CCCGGG, while that of other Fritillaria cirrhosa is CTCGGG. The sequencing results were spliced ​​with double-end data De Novo Assemble and then compared with the reference sequence for key sites. Fig.17 According to the key site comparison of the sequencing results with the standard sequences of Fritillaria cirrhosa and other Fritillaria cirrhosa, only four samples, namely, sample 10, sample 11, sample 12, and sample 13, were Fritillaria cirrhosa, and the other eleven Fritillaria cirrhosa were replaced by other Fritillaria cirrhosa. This result was the same as the result of PCR-RFLP enzyme digestion.

[0264] The sequencing results were subjected to full-length Blast comparison on NCBI, and the results are shown in Table 21. Samples 1 to 9 were identified as Fritillaria cirrhosa, samples 10 to 14 were identified as Fritillaria cirrhosa, and sample 15 was identified as Fritillaria irritans, but the similarity was less than 97%, and it was impossible to determine which type of Fritillaria the sample was.

[0265] Table 21 Blast comparison results with NCBI database

[0266]

[0267]

[0268] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for identifying Fritillaria cirrhosae, characterized in that: It includes at least one primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2, a primer pair consisting of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4, and a primer pair consisting of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.

6.

2. The kit for identifying Fritillaria cirrhosae according to claim 1, characterized in that: The kit comprises a primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.

2.

3. The kit for identifying Fritillaria cirrhosae according to claim 1, characterized in that: The kit comprises a primer pair consisting of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.

4.

4. The kit for identifying Fritillaria cirrhosae according to claim 2 or 3, characterized in that: The kit also includes a PCR buffer, a DNA polymerase and a template DNA, wherein the PCR buffer contains magnesium ions and deoxynucleoside triphosphates.

5. The kit for identifying Fritillaria cirrhosae according to claim 1, characterized in that: The kit comprises a primer pair consisting of an upstream primer shown in SEQ ID NO.5 and a downstream primer shown in SEQ ID NO.

6.

6. The kit for identifying Fritillaria cirrhosae according to claim 5, characterized in that: The kit also includes restriction endonuclease SmaI.

7. The kit for identifying Fritillaria cirrhosae according to claim 6, characterized in that: The kit also comprises a PCR buffer, a DNA polymerase, a template DNA and a restriction endonuclease Sma I; the PCR buffer contains magnesium ions and deoxynucleoside triphosphates.

8. Use of the kit for identifying Fritillaria cirrhosae according to any one of claims 1 to 7 in identifying Fritillaria cirrhosae and its counterfeits.

9. The use according to claim 8, characterized in that: The specific steps include: S1. Extracting total DNA of the sample to be tested; S2. Using the total DNA as template DNA, perform PCR amplification with the above primers, and identify Fritillaria cirrhosa and its counterfeits based on the amplification results.

10. The use according to claim 9, characterized in that: The reaction system of the PCR amplification is: For the primer pair consisting of the upstream primer shown in SEQ ID NO.1 and the downstream primer shown in SEQ ID NO.2, the amplification reaction conditions were: 98°C, 1 min; 98°C, 10 s, 48°C, 15 s, 68°C, 30 s, 40 cycles; 68°C, 5 min; For the primer pair consisting of the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4, the amplification reaction conditions were: 98°C, 1 min; 98°C, 10 s, 62°C, 15 s, 68°C, 30 s, 40 cycles; 68°C, 5 min; For the primer pair consisting of the upstream primer shown in SEQ ID NO.5 and the downstream primer shown in SEQ ID NO.6, the amplification reaction conditions are: 98°C, 1 min; 98°C, 10 s, 45°C, 15 s, 68°C, 30 s, 40 cycles; 68°C, 5 min.

11. The use according to any one of claims 8 to 10, characterized in that: The application is to identify Fritillaria cirrhosa and its counterfeits in traditional Chinese medicines. The kit includes at least one primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2 and a primer pair consisting of an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.

4.

12. The use according to any one of claims 8 to 10, characterized in that: The application includes identifying specific types of counterfeit products, and the kit includes a primer pair consisting of an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2.

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