Primer group, method and kit for identifying authenticity of bulbus fritillariae cirrhosae

Abstract

The invention aims to solve the technical problem of false positive caused by poor primer specificity in the existing bulbus fritillariae cirrhosae true and false identification process, belongs to the technical field of bulbus fritillariae cirrhosae true and false identification, and particularly relates to a bulbus fritillariae cirrhosae true and false identification primer group, method and kit, and the sequence of the primer group is shown as follows: an upstream primer: 5 '-ATATCCTCTCTTTTCTTGGATT-3'; the downstream primer is 5 '-CCGACTTGTGTATCCTCTATTTT-3', and the downstream primer is 5 '- The technical scheme provided by the invention has the technical effects that the authenticity of the bulbus fritillariae cirrhosae can be accurately identified, and false positives are not easily caused in the identification process.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, specifically to drug identification and detection methods, and particularly relates to a primer set, method, and reagent kit for identifying genuine and counterfeit Fritillaria cirrhosa. Background Technology

[0002] Fritillaria cirrhosa is a plant belonging to the Liliaceae family. Fritillaria cirrhosa D.Don, Dark Purple Fritillaria Fritillaria unibracteata Hsiao et KCHsia, Gansu Fritillaria Fritillaria przewalskii Maxim., Fritillaria cirrhosa Fritillaria delavayi Franch., Taibai Fritillaria Fritillaria taipaiensis PY Li or Vabu Fritillaria Fritillaria unibracteata Hsiao et KC Hsia var. wabuensis The dried bulbs of (SY Tanget SC Yue) ZD Liu, S. Wang et SC Chen. Depending on their characteristics, they are commonly known as "Songbei," "Qingbei," "Lubei," and "Cultivated Product." They have the effects of clearing heat and moistening the lungs, resolving phlegm and relieving cough, dispersing nodules and reducing swelling. They are used for dry cough due to lung heat, dry cough with little phlegm, yin deficiency cough, hemoptysis, scrofula, mastitis, and lung abscess.

[0003] The identification methods for Fritillaria cirrhosa in the Chinese Pharmacopoeia include microscopy, thin-layer chromatography, and polymerase chain reaction-restriction endonuclease length polymorphism (PCR-RFLP). The PCR-RFLP method first requires PCR amplification, followed by restriction enzyme digestion of the PCR product, with digestion times ranging from 1 to 2 hours. Furthermore, the pharmacopoeia method is based on the ITS1 sequence. Since this ITS1 sequence is similar to that of some fungi, non-target bands appear in the PCR amplification of Fritillaria cirrhosa contaminated with fungi, interfering with the final result interpretation.

[0004] CZ Wang (Simultaneous identification of Bulbus Fritillariae Cirrhosae using PCR-RFLP analysis), Xu Chuanlin (Study on molecular identification method of Fritillaria cirrhosae [J]. Journal of China Pharmaceutical University, 2010, 41(3):226), and Zhang Wenjuan et al. (Study on the use of polymerase chain reaction-restriction fragment length polymorphism method to detect adulteration of Fritillaria cirrhosae [J]. Journal of Pharmaceutical Analysis, 2014, 34(10):1830) also used the same method as the Chinese Pharmacopoeia.

[0005] CN201910170554.9 relates to a PCR primer and method for identifying the authenticity of Fritillaria cirrhosa or for detecting the degree of adulteration of Fritillaria cirrhosa. Although the technical solution mentions that the two pairs of primers designed can detect the degree of adulteration of Fritillaria cirrhosa, the primers are designed based on the ITS1 region sequence. However, the ITS1 sequence of Fritillaria cirrhosa and its adulterants are very different, with only 1-2 bp differences between the primers. This can easily cause mismatch between the primers and the template, resulting in high background and false positives, which affects the interpretation of the results.

[0006] CN201810133568.9 relates to a rapid PCR method for identifying the authenticity of Fritillaria cirrhosa. In this technical solution, a pair of primers designed based on the ITS1 region sequence is used to identify the authenticity of Fritillaria cirrhosa. However, it also has the problem of false positives due to poor primer specificity.

[0007] CN20181013356.89 utilizes the characteristics of the ITS1 region sequence of Fritillaria cirrhosa, using the 75th base "C" of the ITS1 region as the 3' end, and also introduces a base mismatch at the 3rd base of the 3' end of the primer, which increases the experimental difficulty, easily leads to false positives, and affects the interpretation of results. Summary of the Invention

[0008] This application addresses the aforementioned shortcomings of the prior art by providing a primer combination for identifying genuine and counterfeit Fritillaria cirrhosa, which is less likely to result in false positives during the identification process.

[0009] To solve the above-mentioned technical problems, the technical solution adopted in this application is: a primer set for identifying the authenticity of Fritillaria cirrhosa, the sequence of which is shown below: Upstream primer: 5'-ATATCCTCTTCTCTTTCTTGGATT -3'; Downstream primer: 5'-CCGACTTGTGTATCCTCTATTTT-3'.

[0010] This application also provides a method for distinguishing genuine from counterfeit Fritillaria cirrhosa, the method comprising the following steps: (1) Find a specific DNA sequence that can clearly distinguish Fritillaria cirrhosa from its adulterants (mainly closely related species of Fritillaria cirrhosa); (2) Design specific primer sets based on specific DNA sequences, and then distinguish between genuine and counterfeit Fritillaria cirrhosa by PCR reaction based on the size of PCR product fragments on agarose gel electrophoresis patterns.

[0011] Furthermore, the primer set is a primer set designed based on the specific DNA sequence described below: (1) Common sequence of common adulterants of Fritillaria cirrhosa on chloroplast genome (adulterant-specific sequence): 5'-TCCTCTTCTCTTTCGTTGGATTGGGGGAGAACACATACATAAAAAAATTAGCATGCAATT-3'; (2) Specific sequence of genuine Fritillaria cirrhosa (genuine product specific sequence): 5'-TCCTCTTCTCTTTCGTTGGATTGGAAATTAGCATGCAATT-3'.

[0012] Furthermore, the genuine and counterfeit Fritillaria cirrhosa include six types of origin: Fritillaria cirrhosa, Fritillaria nigra, Fritillaria gansuensis, Fritillaria sambar formosana, Fritillaria taibaiensis, or Fritillaria wabuensis.

[0013] Furthermore, the aforementioned genuine and counterfeit Sichuan fritillary bulbs include six common counterfeit varieties: Ili fritillary bulb, Xinjiang fritillary bulb, Ping fritillary bulb, Zhejiang fritillary bulb, Hubei fritillary bulb, and Anhui fritillary bulb.

[0014] This application also provides a PCR method for identifying genuine and counterfeit Fritillaria cirrhosa. The method includes: extracting genomic DNA from the sample, using the extracted genomic DNA as a template, and conducting a PCR reaction. Based on the size of the PCR product fragments on the agarose gel electrophoresis pattern, the original Fritillaria cirrhosa and common counterfeits are distinguished.

[0015] Furthermore, the PCR reaction system is 20 μL: template DNA (10 ng / μL) 1 μL, primer (10 μmol / L) 0.2 μL, 2×PCR Master Mix 10 μL, sterile ultrapure water 8.6 μL; PCR reaction parameters: 95℃ pre-denaturation for 4 minutes, 30 cycles (95℃ for 30 seconds, 58℃ for 30 seconds, 72℃ for 30 seconds), 72℃ extension for 5 minutes.

[0016] Furthermore, the electrophoresis detection is performed as follows: the gel concentration is 2%, and 1% nucleic acid gel staining agent is added to the gel; electrophoresis is performed for 40-60 minutes under an electric field strength of 4V / cm. After electrophoresis, the gel slide is examined on a gel imaging instrument or an ultraviolet transilluminator.

[0017] This application also provides a PCR kit for identifying genuine and counterfeit Fritillaria cirrhosa, the kit comprising the primer set described above for identifying genuine and counterfeit Fritillaria cirrhosa.

[0018] The advantages and beneficial effects of this application are as follows: 1. Fritillaria-type medicinal materials are easily contaminated by fungi during planting, harvesting, processing, and storage. Current methods generally use primers designed based on ITS1 or ITS sequences. Since the ITS1 or ITS sequences of Fritillaria are homologous to fungi, non-target bands appear in the PCR amplification of Fritillaria cirrhosa contaminated by fungi, which interferes with the final result interpretation. However, this application designs primers based on chloroplast sequences. Fungi do not have chloroplast sequences, so the method of this application will not be affected by fungal contamination in the result interpretation.

[0019] 2. This application can quickly draw conclusions using only one PCR reaction with a single primer pair, while the method included in the Chinese Pharmacopoeia requires enzyme digestion, which not only increases the difficulty of the experiment but also increases the risk of contamination.

[0020] 3. The present application uses a common PCR instrument, that is, by running PCR products on agarose gel electrophoresis and judging the band size, it can achieve the technical effect of determining whether it is Fritillaria cirrhosa or a counterfeit Fritillaria cirrhosa. The instrument and reagent costs are low.

[0021] 4. The primers designed in this application determine authenticity by the length of the amplified product; the primers are completely matched with the sequences of Fritillaria cirrhosa and its common counterfeits, the method is robust, and there are no false positives or false negatives caused by the small base difference between the genuine and counterfeits, thus effectively avoiding false positives and false negatives. Attached Figure Description

[0022] Figure 1 Electrophoresis diagram of Fritillaria cirrhosa identified by the primers described in Example 1 of this application. Detailed Implementation

[0023] The present application is further described in detail below through specific embodiments, but the present application is not limited to the following embodiments.

[0024] This application describes a method for distinguishing genuine from counterfeit Fritillaria cirrhosa, the steps of which include: (1) Find a specific DNA sequence that can clearly distinguish Fritillaria cirrhosa from its adulterants (mainly closely related species of Fritillaria cirrhosa); (2) Design specific primer sets based on specific DNA sequences, and then distinguish between genuine and counterfeit Fritillaria cirrhosa by PCR reaction based on the size of PCR product fragments on agarose gel electrophoresis patterns.

[0025] The primer set described in this application is a primer set designed based on the specific DNA sequence described below: (1) Common sequence of common adulterants of Fritillaria cirrhosa on chloroplast genome (adulterant-specific sequence): 5'-TCCTCTTCTCTTTCGTTGGATTGGGGGAGAACACATACATAAAAAAATTAGCATGCAATT-3'; (2) Specific sequence of genuine Fritillaria cirrhosa (genuine product specific sequence): 5'-TCCTCTTCTCTTTCGTTGGATTGGAAATTAGCATGCAATT-3'.

[0026] This application designs a primer set based on the specific DNA sequence described above. This primer set includes the following primer set: Upstream primer: 5'-ATATCCTCTTCTCTTTCTTGGATT -3'; Downstream primer: 5'-CCGACTTGTGTATCCTCTATTTT-3'.

[0027] Specifically, this application refers to the following comparative sequence of Fritillaria cirrhosa and common adulterants: Fritillaria TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Dark purple Fritillaria TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Gansu Fritillaria TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Fritillaria TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Wabu Fritillaria TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Taibai Fritillaria TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Fritillaria thunbergii Miq. var. chekiangensis Hsiao & K. C. Hsia TTATTATATA CTAATGGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Fritillaria thunbergii Miq. TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Fritillaria walujewii Regel TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Fritillaria pallidiflora Schrenk TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Fritillaria hupehensis Hsiao & K. C. Hsia TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Fritillaria anhuiensis Hsiao & K. C. Hsia TTATTATATA CTAATAGATT TAATTAATAT TAATTATTAA TATAATTAATTAATATAATA

[28840] Fritillaria cirrhosa D. Don TCCTCTTCTC TTTC-TTGGA TTGGG----- ---------- ------------------TT

[28900] Fritillaria unibracteata Hsiao & K. C. Hsia TCCTCTTCTC TTTC-TTGGA TTGGG----- ---------- ------------------TT

[28900] Fritillaria przewalskii Maxim. TCCTCTTCTC TTTC-TTGGA TTGGG----- ---------- ------------------TT

[28900] Fritillaria delavayi Franch. TCCTCTTCTC TTTC-TTGGA TTGGG----- ---------- ------------------TT

[28900] Fritillaria unibracteata Hsiao & K. C. Hsia var. wabuensis S. Y. Tang & S. C. Yue TCCTCTTCTC TTTC-TTGGA TTGGG----- ---------- ------------------TT

[28900] Taibai Fritillaria thunbergii TCCTCTTCTC TTTC-TTGGA TTGGG----- ---------- ------------------TT

[28900] Fritillaria TCCTCTTCTC TTTCGTTGGA TTGGGGGAGA ACACATACAT AAAAAAATTAGCATGCAATT

[28900] Fritillaria TCCTCTTCTC TTTCGTTGGA TTGGGGGAGA ACACATACAT AAAAAAATTAGCATGCAATT

[28900] Xinjiang Fritillaria cirrhosa TCCTCTTCTC TTTCGTTGGA TTGGGGGAGA ACACATACAT AAAAAA-TTAGCATGCAATT

[28900] Ili Fritillaria cirrhosa TCCTCTTCTC TTTCGTTGGA TTGGGGGAGA ACACATACAT AAAAAA-TTAGCATGCAATT

[28900] Hubei Fritillaria cirrhosa TCCTCTTCTC TTTCGTTGGA TTGGGGGAGA ACACATACAT AAAAAA-TTAGCATGCAATT

[28900] Fritillaria TCCTCTTCTC TTTCGTTGGA TTGGGGGAGA ACACATACAT AAAAAA-TTAGCATGCAATT

[28900] Fritillaria TGAATGAAT AGATTTTTTT AATTAAAATA TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] Dark purple mother-of-pearl TGATGAGAT AGATTTTTTT AATTAAAA-A TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] Gansu Fritillaria cirrhosa TGATGAGAT AGATTTTTTT AATTAAAATA TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] Fritillaria TGAATGAGAT AGATTTTTTT AATTAAAATA TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] TGAATGAGAT AGATTTTTTT AATTAAAA-A TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] Taibai Fritillaria TGAATGAGAT AGATTTTTTT AATTAAAA-A TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] Fritillaria TGAATGAGAT AGAATTTTTT AATTAAAA-A TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] Fritillaria TGAATGAAT AGAATTTTTT AATTAAAA-A TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] Xinjiang Fritillaria TGAATGAGAT AGAATTTTTT AATTAAAA-A TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] Ili Fritillaria thunbergii TGATGAGAT AGAATTTTTT AATTAAAA-A TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] Hubei Fritillaria TGAATGAGAT AGAATTTTTT AATTAAAA-A TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] Fritillaria TGAATGAGAT AGAATTTTTT AATTAAAA-A TAGAGGATAC ACAAGTCGGAGTTGGAAAAG

[28960] The reaction conditions, including primer concentration and annealing temperature, were optimized. The optimized PCR reaction system consisted of 20 μL: 1 μL template DNA (10 ng / μL), 0.2 μL primers (10 μmol / L), 10 μL 2×PCR Master Mix, and 8.6 μL sterile ultrapure water. The PCR reaction parameters were: 95℃ pre-denaturation for 4 minutes, 30 cycles (95℃ for 30 seconds, 58℃ for 30 seconds, 72℃ for 30 seconds), and 72℃ extension for 5 minutes.

[0028] Electrophoresis detection: The gel concentration is 2%, and 1% nucleic acid gel staining agent is added to the gel; electrophoresis is performed for 40-60 minutes under an electric field strength of 4V / cm. After electrophoresis, the gel slides are examined on a gel imaging system or a UV transilluminator.

[0029] The results are interpreted as follows: if the test sample contains a 50-100 bp fragment, it is Fritillaria cirrhosa; if the test sample contains a 100-150 bp fragment, it is a counterfeit Fritillaria cirrhosa; if none of the above fragments are present, it is a counterfeit Fritillaria cirrhosa. This is because, based on the comparison sequences of Fritillaria cirrhosa and common counterfeits in this application, genuine Fritillaria cirrhosa has a 33 bp deletion mutation, which results in the PCR product of genuine Fritillaria cirrhosa being 33 bp shorter than that of the counterfeit. It is easy to distinguish between genuine and counterfeit products by the size of the PCR product.

[0030] Example: Take 0.1g of this product, wash successively with 1ml of 75% ethanol and 1ml of sterile ultrapure water, blot dry the surface moisture, and grind into an extremely fine powder in a mortar. Take 20mg, place in a 1.5ml centrifuge tube, and extract DNA using a plant genomic DNA rapid extraction kit [add 400μl of buffer AP1 and 4μl of RNase solution (10mg / ml), vortex, heat in a 65℃ water bath for 10 minutes, add 130μl of buffer AP2, mix thoroughly, cool in an ice bath for 5 minutes, centrifuge (14000 rpm) for 10 minutes; transfer the supernatant to another centrifuge tube, add 1.5 times the volume of buffer AP3, mix well, add to the adsorption column, centrifuge (13000 rpm) for 1 minute, discard the filtrate, add 700μl of wash buffer, centrifuge (12000 rpm) for 30 seconds, discard the filtrate; add another 500μl of wash buffer, centrifuge (12000 rpm) for 30 seconds, discard the filtrate; then add ... Centrifuge at 12,000 rpm for 30 seconds, discard the filtrate; then centrifuge at 13,000 rpm for 2 minutes, remove the adsorption column, place it in another centrifuge tube, add 50 μl of elution buffer, incubate at room temperature for 3-5 minutes, centrifuge at 12,000 rpm for 1 minute, add the elution buffer back into the adsorption column, incubate at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute, take the elution buffer as the test solution, and store it in a 4°C refrigerator for later use.

[0031] PCR reaction identification primers: This primer set includes the following primer sets: Upstream primer: 5'-ATATCCTCTTCTCTTTCTTGGATT -3'; Downstream primer: 5'-CCGACTTGTGTATCCTCTATTTT-3'.

[0032] PCR reaction system: Performed in 200 μl centrifuge tubes, with a total reaction volume of 20 μl: 1 μL template DNA, 0.2 μL primers (10 μmol / L), 10 μL 2×PCR Master Mix, and 8.6 μL sterile ultrapure water. Place the centrifuge tubes in a PCR instrument. PCR reaction parameters: 95℃ pre-denaturation for 4 minutes, 30 cycles (95℃ for 30 seconds, 58℃ for 30 seconds, 72℃ for 30 seconds), and 72℃ extension for 5 minutes.

[0033] Agarose gel electrophoresis: The gel concentration is 2%, and 1% nucleic acid gel staining agent is added to the gel. Electrophoresis is performed at an electric field strength of 4V / cm for 40-60 minutes. After electrophoresis, the gel slides are examined on a gel imaging system or a UV transilluminator.

[0034] Figure 1 This is an electrophoresis diagram of identification using the primers described in this application; from the diagram, lane M1 is a 50 bp DNA ladder, lane M2 is DNA Marker I, lanes 1-6 are genuine Fritillaria cirrhosa, namely Fritillaria cirrhosa, Fritillaria thunbergii, Fritillaria thunbergii, Fritillaria taibaiensis, Fritillaria gansuensis, and Fritillaria wabuensis, respectively, and lanes 7-12 are counterfeit Fritillaria cirrhosa, namely Fritillaria pingseng, Fritillaria xinjiangensis, Fritillaria yiliensis, Fritillaria thunbergii, Fritillaria huinanensis, and Fritillaria hubeiensis, respectively.

[0035] Conclusion: Genuine Fritillaria cirrhosa showed a unique band in its PCR product between 50-100 bp, while counterfeit Fritillaria cirrhosa showed a unique band between 100-150 bp. The size of the PCR product bands allows for rapid identification of genuine and counterfeit Fritillaria cirrhosa.

[0036] Table 1 Sample Information Table Sample Name Latin name Source / Origin The method of this application determines the result. Determination of results according to the method in the Chinese Pharmacopoeia Fritillaria cirrhosa D. Don Sichuan Lotus Pond Medicinal Herbs Market Authentic Fritillaria cirrhosa Authentic Fritillaria cirrhosa Dark purple mother-of-pearl Hsiao et KCHsia Sichuan Authentic Fritillaria cirrhosa Authentic Fritillaria cirrhosa Gansu Fritillaria Maxim. Gansu Province Authentic Fritillaria cirrhosa Authentic Fritillaria cirrhosa Fritillaria thunbergii Franch. Maoxian County, Sichuan Authentic Fritillaria cirrhosa Authentic Fritillaria cirrhosa Taibai Fritillaria PYLi Chengkou, Sichuan Vabu fritillary Hsiao et KCHsia var.(SY Tang et SC Yue) ZD Liu, S.Wanget SC Chen Authentic Fritillaria cirrhosa Authentic Fritillaria cirrhosa Fritillaria Maxim. Tonghua, Jilin counterfeit Fritillaria cirrhosa counterfeit Fritillaria cirrhosa Ili Fritillaria Schrenk Yili, Xinjiang counterfeit Fritillaria cirrhosa counterfeit Fritillaria cirrhosa Xinjiang Fritillaria Regel Xinjiang counterfeit Fritillaria cirrhosa counterfeit Fritillaria cirrhosa Fritillaria thunbergii Miq. Ningbo, Zhejiang counterfeit Fritillaria cirrhosa counterfeit Fritillaria cirrhosa Hubei Fritillaria Hsiao et KCHsia Enshi, Hubei counterfeit Fritillaria cirrhosa counterfeit Fritillaria cirrhosa Fritillaria cirrhosa SCChen etS.P.Yin Anhui Bozhou Medicinal Herbs Market counterfeit Fritillaria cirrhosa counterfeit Fritillaria cirrhosa All the above samples were confirmed using the PCR-RFLP method specified in the Chinese Pharmacopoeia.

[0037] Primers used: Upstream primer: 5'-CGTAACAAGGTTTCCGTAGGTGAA-3' Downstream primer: 5'-GCTACGTTCTTCATCGAT-3'; The primer here is the one used in the Chinese Pharmacopoeia to identify Fritillaria cirrhosa, and is used to verify the primer in this application.

[0038] As can be seen from the above specific embodiments, the method of this application can find specific DNA sequences that can clearly distinguish Fritillaria cirrhosa from its adulterants (mainly closely related species of the Fritillaria genus); design specific primers, and through PCR reaction, based on the size of the PCR product fragments on the agarose gel electrophoresis pattern, distinguish between six original Fritillaria cirrhosa species (Fritillaria cirrhosa, Fritillaria purpurea, Fritillaria gansuensis, Fritillaria spathulata, Fritillaria taibaiensis or Fritillaria wabuensis) and six common adulterants (Fritillaria yiliensis, Fritillaria xinjiangensis, Fritillaria pingseng, Fritillaria thunbergii, Fritillaria hupehensis, Fritillaria huishensis, Fritillaria huangshensis, Fritillaria hui ...

Claims

1. A primer set for identifying genuine and counterfeit Fritillaria cirrhosa, characterized in that: The primer sequence is shown below: Upstream primer: 5'-ATATCCTCTTCTCTTTCTTGGATT -3'; Downstream primer: 5'-CCGACTTGTGTATCCTCTATTTT-3'.

2. A method for distinguishing genuine from counterfeit Fritillaria cirrhosa, characterized in that: The steps of this method include: (1) Find a specific DNA sequence that can clearly distinguish Fritillaria cirrhosa from its adulterants; (2) Design specific primer sets based on specific DNA sequences, and then distinguish between genuine and counterfeit Fritillaria cirrhosa by PCR reaction based on the size of PCR product fragments on agarose gel electrophoresis patterns.

3. The method for distinguishing genuine from counterfeit Fritillaria cirrhosa according to claim 2, characterized in that: The primer set described above is a primer set designed based on the specific DNA sequence described below: (1) Common sequence of common adulterants of Fritillaria cirrhosa on chloroplast genome, i.e. adulterant-specific sequence: 5'-TCCTCTTCTCTTTCGTTGGATTGGGGGAGAACACATACATAAAAAAATTAGCATGCAATT-3'; (2) The specific sequence of genuine Fritillaria cirrhosa is: 5'-TCCTCTTCTCTTTCGTTGGATTGGAAATTAGCATGCAATT-3'.

4. The method for distinguishing genuine from counterfeit Fritillaria cirrhosa according to claim 2, characterized in that: The genuine and counterfeit Fritillaria cirrhosa mentioned include six types of origin: Fritillaria cirrhosa, Fritillaria purpurea, Fritillaria gansuense, Fritillaria spathulata, Fritillaria taibaiense, or Fritillaria wabu.

5. The method for distinguishing genuine from counterfeit Fritillaria cirrhosa according to claim 4, characterized in that: The aforementioned genuine and counterfeit Sichuan fritillary bulbs include six common counterfeit varieties: Ili fritillary bulb, Xinjiang fritillary bulb, Ping fritillary bulb, Zhejiang fritillary bulb, Hubei fritillary bulb, and Anhui fritillary bulb.

6. A PCR method for identifying genuine and counterfeit Fritillaria cirrhosa, characterized in that: The method includes: extracting genomic DNA from the sample, using the extracted genomic DNA as a template, and conducting a PCR reaction. Based on the size of the PCR product fragments on the agarose gel electrophoresis pattern, the original Fritillaria cirrhosa and common adulterants are distinguished.

7. The PCR method for identifying the authenticity of Fritillaria cirrhosa according to claim 6, characterized in that: The PCR reaction system consisted of 20 μL: 1 μL template DNA (10 ng / μL); 0.2 μL primer (10 μmol / L); 10 μL 2×PCR Master Mix; and 8.6 μL sterile ultrapure water. The PCR reaction parameters were: 95℃ pre-denaturation for 4 minutes; 30 cycles of 95℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 30 seconds; and extension at 72℃ for 5 minutes.

8. The PCR method for identifying the authenticity of Fritillaria cirrhosa according to claim 6, characterized in that: The detection procedure based on agarose gel electrophoresis is as follows: the gel concentration is 2%, and 1% nucleic acid gel staining agent is added to the gel; electrophoresis is performed for 40-60 minutes under an electric field strength of 4V / cm. After electrophoresis, the gel slide is examined on a gel imaging instrument or an ultraviolet transilluminator.

9. A PCR kit for identifying genuine and counterfeit Fritillaria cirrhosa, characterized in that: The kit includes the primer set for identifying genuine and counterfeit Fritillaria cirrhosa as described in claim 1.

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