Chrysanthemum nankingense specific detection primer, identification method and application

Abstract

The invention discloses a chrysanthemum nankingense specific detection primer, an identification method and application, and the chrysanthemum nankingense specific detection primer comprises a forward primer JHN-1F: 5 '-ATGACGGAATCAATATC ACTGG-3', a reverse primer JHN-1R: 5 '-GACTTTCCAGTTGCACGTGG-3', a reverse primer JHN-1R: 5 '- The invention also provides a chrysanthemum nankingense PCR electrophoresis detection and identification method and a fluorescent quantitative PCR detection and identification method. According to the present invention, with the forward primer JHN-1F and the reverse primer JHN-1R, the chrysanthemum nankingense can be accurately identified from the commercially available chrysanthemum indicum, and the identification can be performed through the common PCR method and the fluorescent quantitative PCR method; based on the method, chrysanthemum indicum and chrysanthemum nankingense which cannot be identified by DNA bar codes can be identified.

Description

Technical Field

[0001] The present invention relates to the technical field of chrysanthemum brain variety identification, and specifically relates to a chrysanthemum brain specific detection primer, an identification method and an application. Background Art

[0002] Wild chrysanthemum and chrysanthemum brain are recorded as one species in the Flora of China, but in actual application, wild chrysanthemum has medicinal value such as clearing away heat and detoxification, while chrysanthemum brain flower is mainly used for edible value such as cooling and improving eyesight. In actual production and life, the two are sold as two species with different effects. Due to the high medicinal value of wild chrysanthemum, its price is relatively high; while the price of chrysanthemum brain flower is relatively low, so it is common to see chrysanthemum brain flower sold as wild chrysanthemum in the market, disrupting the market of wild chrysanthemum and causing harm to the health of patients.

[0003] Since the two have similar characteristics, it is difficult to distinguish them using traditional identification methods. For example, DNA barcodes such as ITS2, matk, and rbcL cannot distinguish them, indicating that the two are closely related. Therefore, how to accurately identify chrysanthemum brain becomes a technical problem that needs to be solved in this application. Summary of the invention

[0004] The purpose of the present invention is to provide a chrysanthemum brain-specific detection primer, identification method and application to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A chrysanthemum brain specific detection primer, comprising:

[0007] Forward primer JHN-1F: 5′-ATGACGGAATCAATATCACTGG-3′;

[0008] Reverse primer JHN-1R: 5′-GACTTTCTTCAGTTGCACGTGG-3′.

[0009] A method for identifying chrysanthemum brain, specifically PCR electrophoresis detection, comprising the following steps:

[0010] S1, extracting DNA of the species to be tested;

[0011] S2, using the DNA extracted in step S1 as an amplified DNA template, and using the forward primer JHN-1F and the reverse primer JHN-1R described in claim 1 as amplification primers to perform PCR amplification;

[0012] S3. Perform agarose gel electrophoresis on the amplified product of step S2. If a band is produced, the variety to be tested is chrysanthemum brain, otherwise it is not.

[0013] As a further specific scheme of the present invention, the PCR reaction system in step S2 is 25 μL, specifically including: 1 μL of forward primer JHN-1F, 1 μL of reverse primer JHN-1R, 1 μL of template amount of amplified DNA template, 12.5 μL of PCR MIX, and 9.5 μL of ddH2O;

[0014] The PCR amplification program was as follows: initial denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 1 min, 35 cycles; and final extension at 72°C for 5 min.

[0015] A method for identifying chrysanthemum brain, specifically a fluorescence quantitative PCR detection, comprising the following steps:

[0016] H1. Extract DNA of the species to be tested;

[0017] H2. Using the DNA extracted in step H1 as an amplification template, and using the forward primer JHN-1F and the reverse primer JHN-1R described in claim 1 as amplification primers, perform fluorescence quantitative PCR amplification. If fluorescence is generated, the variety to be tested is chrysanthemum brain, otherwise it is not;

[0018] As a further specific scheme of the present invention, the total reaction system of the fluorescent quantitative PCR in step H2 is 20 μL, specifically including: 2 μL of amplified DNA template, 0.4 μL of forward primer JHN-1F, 0.4 μL of reverse primer JHN-1R, 10 μL of SYBRmix dye, and 7.2 μL of ddH2O;

[0019] The fluorescence quantitative PCR amplification program was as follows: initial denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 65°C for 20 s, and extension at 72°C for 25 s, for 40 cycles; denaturation at 95°C for 30 s, renaturation at 65°C for 30 s, and gradual denaturation from 65 to 95°C, with the temperature rising to 95°C at a rate of 0.5°C / s.

[0020] As a further specific solution of the present invention, the specific steps of extracting the DNA of the species to be tested in step S1 or step H1 are as follows:

[0021] Take 50 mg of the flower of the tested variety, wipe the surface with 75% ethanol, evaporate the ethanol, add liquid nitrogen to grind in a mortar, and extract the sample DNA using the CTAB method.

[0022] The application of the aforementioned chrysanthemum brain-specific detection primers in identifying chrysanthemum brain varieties.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: the forward primer JHN-1F and the reverse primer JHN-1R designed by the present invention can accurately identify chrysanthemum oleoside from commercially available wild chrysanthemum, and can be identified by both common PCR and fluorescent quantitative PCR methods. Based on this method, wild chrysanthemum and chrysanthemum oleoside that cannot be identified by DNA barcodes can be identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the result of PCR electrophoresis of sample DNA using universal primer ITS2;

[0025] Figure 2 This is the electrophoresis result diagram of PCR electrophoresis specific primer verification;

[0026] Figure 3 This is the PCR electrophoresis result of wild chrysanthemum and chrysanthemum brain samples from other regions;

[0027] Figure 4 The figure shows the PCR electrophoresis results of commercially available wild chrysanthemum and chrysanthemum brain samples;

[0028] Figure 5 The results of fluorescence quantitative PCR of DNA samples from Chrysanthemum indica and Chrysanthemum chinense;

[0029] Figure 6 This is the result diagram of the investigation of the detection limit of fluorescent quantitative PCR;

[0030] Figure 7 This is the result of fluorescence quantitative PCR using commercially available wild chrysanthemum sample DNA and specific primers. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] 1 Materials

[0033] 1.1 Sample

[0034] The samples were collected in Anhui (Lu'an Mazongling National Nature Reserve, Huangshan, Wuhu) and Jiangsu (Nanjing, Zhenjiang). The whole wild chrysanthemum and chrysanthemum brain were identified by Chinese medicine practitioners as wild chrysanthemum and chrysanthemum brain. 9 and 5 batches of wild chrysanthemum and chrysanthemum brain were purchased from the Bozhou medicinal materials trading market, respectively. All collected samples were stored in the cold storage of Bozhou College. See Table 1 for details.

[0035] Table 1: Sample information

[0036] serial number Sample name Latin name source Location YJH-1 Wild Chrysanthemum C.IndiciFlos Mazongling, Lu'an, Anhui Flowers YJH-2 Wild Chrysanthemum C.IndiciFlos Anhui Huangshan Flowers YJH-3 Wild Chrysanthemum C.IndiciFlos Wuhu, Anhui Flowers JHN-1 Chrysanthemum brain C.nankingense Anhui Bozhou Flowers JHN-2 Chrysanthemum brain C.nankingense Nanjing, Jiangsu Flowers JHN-3 Chrysanthemum brain C.nankingense Zhenjiang, Jiangsu Flowers

[0037] 1.2 Reagents

[0038] The reagents used were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. and Shanghai Leibus Technology Co., Ltd., respectively. See Table 2 for details.

[0039] Table 2: Reagent names and manufacturers

[0040] Reagent name Manufacturer Plant Genomic DNA Extraction Kit Shanghai Bioengineering Co., Ltd. Agarose1110GR100 Shanghai Leibusi Technology Co., Ltd. TBEBufferPremixedPowder Shanghai Bioengineering Co., Ltd. 2×TaqMasterMix Shanghai Bioengineering Co., Ltd. DNA molecular weight standard Marker (100-2000bp) Shanghai Bioengineering Co., Ltd. GelRed Shanghai Bioengineering Co., Ltd.

[0041] 1.3 Instruments

[0042] Table 3: Equipment name and manufacturer

[0043]

[0044]

[0045] 2 Methods

[0046] 2.1 DNA extraction

[0047] Take 50 mg of wild chrysanthemum and chrysanthemum brain flowers, wipe the surface with 75% ethanol, evaporate the ethanol, add liquid nitrogen to a mortar and grind, extract the sample DNA using the CTAB method, and determine the nucleic acid concentration and A260 / 280 using an ultra-trace nucleic acid protein analyzer. The nucleic acid concentration of wild chrysanthemum is 110-120 ng / μL, and the A260 / 280 ratio is between 1.8 and 2.0. The nucleic acid concentration of chrysanthemum brain is 260-280 ng / μL, and the A260 / 280 ratio is between 1.8 and 2.0.

[0048] The extracted sample DNA was amplified by PCR using universal primer ITS2 and then electrophoresed. For details, see Figure 1 ,exist Figure 1 Middle: M is Marker DL2000, 1 is wild chrysanthemum, 2 is chrysanthemum brain; Figure 1 It can be seen that the bands of wild chrysanthemum and chrysanthemum brain are bright and have no tailing phenomenon, indicating that the extracted sample DNA meets the requirements of the next PCR experiment.

[0049] Among them, the universal primer ITS2 includes:

[0050] Forward primer ITS-F5a: 5′-CCTTATCATTTAGAGGAAGGAG-3′;

[0051] Reverse primer ITS-R5a: 5′-TCCTCCGCTTATTGATATGC-3′.

[0052] 2.2 PCR electrophoresis detection

[0053] The extracted sample DNA was used as an amplified DNA template, and PCR amplification was performed using forward primer JHN-1F and reverse primer JHN-1R as amplification primers; the amplified product was electrophoresed in a 2% agarose gel under the conditions of 150V, 400mA, and 35min.

[0054] The PCR reaction system is 25 μL, including: 1 μL of forward primer JHN-1F, 1 μL of reverse primer JHN-1R, 1 μL of amplified DNA template, 12.5 μL of PCR MIX, and 9.5 μL of ddH2O;

[0055] The PCR amplification program was as follows: initial denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 1 min, 35 cycles; and final extension at 72°C for 5 min.

[0056] 2.3. PCR electrophoresis specific primer verification

[0057] In the annealing temperature range of 60℃~65℃, five gradients were selected for PCR amplification. Figure 2 ,exist Figure 2 In: M is Marker DL2000, serial numbers 1, 3, 5, 7, 9, 11, 13, 15 represent wild chrysanthemum; serial numbers 2, 4, 6, 8, 10, 12, 14, 16 represent chrysanthemum; the annealing temperature of serial numbers 1 and 2 is 60°C, the annealing temperature of serial numbers 3 and 4 is 60.6°C, the annealing temperature of serial numbers 5 and 6 is 62.2°C, the annealing temperature of serial numbers 7 and 8 is 64.3°C, and the annealing temperature of serial numbers 9 and 10 is 65°C; Figure 2 It can be seen that when the annealing temperature is 60-64.3℃, the PCR products of both wild chrysanthemum and chrysanthemum brain produce bands, but the base length of the wild chrysanthemum amplification product is slightly longer than that of chrysanthemum brain; when the annealing temperature is 65℃, the PCR product only has a band of chrysanthemum brain while wild chrysanthemum has no band; this indicates that at the annealing temperature of 65℃, the designed specific primers are specific and can identify chrysanthemum brain from wild chrysanthemum.

[0058] 2.4. Investigation of the suitability of PCR electrophoresis

[0059] Wild chrysanthemum and chrysanthemum brain samples collected from other regions were selected, and the sample DNA was extracted and amplified by PCR with specific primers and electrophoresed. The annealing temperature of PCR amplification was 65°C. For details, see Figure 3 ,exist Figure 3 Middle: M is MarkerDL2000, 1 is wild chrysanthemum, 2 is chrysanthemum brain; Figure 3It can be seen that all samples of Chrysanthemum chinense amplified bands, while no bands were produced in Chrysanthemum indica, which further demonstrated that the designed specific primers were specific after PCR reaction at an annealing temperature of 65°C.

[0060] PCR amplification and electrophoresis were performed using the specific primers of chrysanthemum brain and the DNA samples of commercial wild chrysanthemum and chrysanthemum brain, respectively. Figure 4 ,exist Figure 4 In the middle: M is Marker DL2000, 1-9 are commercially available wild chrysanthemum samples, 10-14 are commercially available chrysanthemum brain samples; Figure 4 It can be seen that samples 2, 8 and 9 showed chrysanthemum brain-specific bands, indicating that the three batches of samples are chrysanthemum brain; samples 10 to 14 all showed chrysanthemum brain-specific bands, indicating that chrysanthemum brain is being sold in the market as wild chrysanthemum.

[0061] 2.5. Fluorescence quantitative PCR detection

[0062] The extracted sample DNA was used as an amplification template, and the forward primer JHN-1F and the reverse primer JHN-1R were used as amplification primers to perform fluorescence quantitative PCR amplification detection;

[0063] The total reaction system of fluorescent quantitative PCR was 20 μL, including: 2 μL amplified DNA template, 0.4 μL forward primer JHN-1F, 0.4 μL reverse primer JHN-1R, 10 μL SYBR Mix dye, and 7.2 μL ddH2O;

[0064] The fluorescence quantitative PCR amplification program was as follows: initial denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 65°C for 20 s, and extension at 72°C for 25 s, for 40 cycles; denaturation at 95°C for 30 s, renaturation at 65°C for 30 s, and gradual denaturation from 65 to 95°C, with the temperature rising to 95°C at a rate of 0.5°C / s.

[0065] 2.6. Verification of specific primers for fluorescent quantitative PCR

[0066] DNA samples of wild chrysanthemum and chrysanthemum brain were amplified by fluorescence quantitative PCR with chrysanthemum brain specific primers. Figure 5 ,exist Figure 5 In: A is chrysanthemum brain, B is wild chrysanthemum; Figure 5 It can be seen that chrysanthemum brain produces fluorescent signals, and its melting curve Tm value is 77°C, while wild chrysanthemum has no fluorescent signal, indicating that the designed specific primers are specific.

[0067] 2.7. Investigation of detection limit of fluorescent quantitative PCR

[0068] The concentration of the amplified DNA template was shown in Table 4. The concentration was diluted 4 times by 10 times to investigate the detection limit of the template concentration. The initial concentration of the sample DNA of chrysanthemum brain was 5×10 4 pg / μL.

[0069] Table 4: Fluorescence quantification detection limits

[0070] Dilution multiple 100％ <![CDATA[10 -1 ]]> <![CDATA[10 -2 ]]> <![CDATA[10 -3 ]]> <![CDATA[10 -4 ]]> Sample template amount (pg / μL) <![CDATA[5×10 4 ]]> <![CDATA[5×10 3 ]]> <![CDATA[5×10 2 ]]> 50 5

[0071] The detection limit of chrysanthemum brain was investigated, and the DNA template of chrysanthemum brain was amplified by fluorescence quantitative PCR from 5pg / μL to 50ng / μL. Figure 6 ,exist Figure 6 Middle: 1-5 corresponds to a DNA template concentration of 5×10 4 pg / μL, 5×10 3 pg / μL, 5×10 2 pg / μL, 50pg / μL, 5pg / μL; by Figure 6 It can be seen that when the DNA template concentration of chrysanthemum brain is as low as 5pg / μL, fluorescence signals are still generated, and its CT value is less than 35, indicating that chrysanthemum brain-specific primers can detect chrysanthemum brain DNA samples as low as 5pg / μL.

[0072] 2.8. Applicability of Fluorescence Quantitative PCR

[0073] The commercially available wild chrysanthemum sample DNA was amplified by fluorescent quantitative PCR with chrysanthemum brain-specific primers, and a melting curve analysis was performed. For details, see Figure 7 ,exist Figure 7 In: A is 2, 8, 9 wild chrysanthemum samples, B is 1, 3, 4, 5, 6, 7 wild chrysanthemum samples. Figure 7 It can be seen that wild chrysanthemum samples 2, 8, and 9 have fluorescent signals and their melting curves are 77°C, while other samples have no fluorescent signals, indicating that wild chrysanthemum samples 2, 8, and 9 should be chrysanthemum brain.

[0074] The specificity, reproducibility, detection limit and applicability of the forward primer JHN-1F and the reverse primer JHN-1R were investigated in the embodiment of the present invention. Amplification and verification were performed by conventional PCR and fluorescence quantitative PCR, and the designed chrysanthemum brain-specific primers were able to specifically identify chrysanthemum brain, indicating that the primer specificity was good. By verifying 9 batches of wild chrysanthemums available on the market, conventional PCR and fluorescence quantitative PCR based on specific primers were able to indicate that 3 batches were chrysanthemum brains, indicating that 1 / 3 of the wild chrysanthemum samples collected on the market were chrysanthemum brains.

[0075] The forward primer JHN-1F and the reverse primer JHN-1R designed by the present invention can accurately identify chrysanthemum oleoside from commercially available wild chrysanthemum, and can be identified by both common PCR and fluorescent quantitative PCR methods. Based on this method, wild chrysanthemum and chrysanthemum oleoside that cannot be identified by DNA barcodes can be identified.

Claims

1. A primer for detecting chrysanthemum brain-specificity, characterized in that: include: Forward primer JHN-1F: 5′-ATGACGGAATCAATATCACTGG-3′; Reverse primer JHN-1R: 5′-GACTTTCTTCAGTTGCACGTGG-3′.

2. A method for identifying chrysanthemum brain, characterized in that: Specifically, the PCR electrophoresis detection includes the following steps: S1, extracting DNA of the species to be tested; S2, using the DNA extracted in step S1 as an amplified DNA template, and using the forward primer JHN-1F and the reverse primer JHN-1R described in claim 1 as amplification primers to perform PCR amplification; S3. Perform agarose gel electrophoresis on the amplified product of step S2. If a band is produced, the variety to be tested is chrysanthemum brain, otherwise it is not.

3. The method for identifying chrysanthemum brain according to claim 2, characterized in that: The PCR reaction system in step S2 is 25 μL, specifically including: 1 μL of forward primer JHN-1F, 1 μL of reverse primer JHN-1R, 1 μL of template amount of amplified DNA template, 12.5 μL of PCR MIX, and 9.5 μL of ddH2O; The PCR amplification program was as follows: initial denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 1 min, 35 cycles; and final extension at 72°C for 5 min.

4. A method for identifying chrysanthemum brain, characterized in that: Specifically, the fluorescence quantitative PCR detection includes the following steps: H1. Extract DNA of the species to be tested; H2. Use the DNA extracted in step H1 as an amplification template, and use the forward primer JHN-1F and the reverse primer JHN-1R described in claim 1 as amplification primers to perform fluorescence quantitative PCR amplification. If fluorescence is generated, the variety to be tested is chrysanthemum brain, otherwise it is not.

5. The method for identifying chrysanthemum brain according to claim 4, characterized in that: The total reaction system of the fluorescent quantitative PCR in step H2 is 20 μL, specifically including: 2 μL of amplified DNA template, 0.4 μL of forward primer JHN-1F, 0.4 μL of reverse primer JHN-1R, 10 μL of SYBR Mix dye, and 7.2 μL of ddH2O; The fluorescence quantitative PCR amplification program was as follows: initial denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 65°C for 20 s, and extension at 72°C for 25 s, for 40 cycles; denaturation at 95°C for 30 s, renaturation at 65°C for 30 s, and gradual denaturation from 65 to 95°C, with the temperature rising to 95°C at a rate of 0.5°C / s.

6. A method for identifying chrysanthemum brain according to any one of claims 2 to 4, characterized in that: The specific steps of extracting DNA of the species to be tested in step S1 or step H1 are as follows: Take 50 mg of the flower of the tested variety, wipe the surface with 75% ethanol, evaporate the ethanol, add liquid nitrogen to grind in a mortar, and extract the sample DNA using the CTAB method.

7. Use of the chrysanthemum brain-specific detection primers described in claim 1 in identifying chrysanthemum brain varieties.

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