Pseudo-ginseng component rapid detection method based on Proofman-LMTIA and application

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a pseudo-ginseng component rapid detection method based on a Proofman-LMTIA technology. According to the method, a pseudo-ginseng ITS2 sequence is taken as a specific target, and high-specificity and high-sensitivity detection of pseudo-ginseng components is realized through a designed LMTIA primer and a Proofman probe. Compared with the traditional detection technology, the invention has the advantages of simplified primer design, low-cost equipment requirement, rapid detection (only about 30 minutes) and wide application range. The method is not only suitable for detection of raw materials such as pseudo-ginseng seeds, seedlings, roots and rhizomes, decoction pieces and the like, but also can be used for authenticity identification of terminal products such as food, medicines, health care products and the like containing pseudo-ginseng components. Through innovative application of the Proofman-LMTIA technology, an effective solution is provided for rapid and accurate identification of pseudo-ginseng and derivative products thereof, and the method has important market application prospects and social benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and specifically relates to a method for rapid detection of notoginseng components based on Proofman-LMTIA with a notoginseng ITS2 sequence as a target, a primer set, a probe and applications thereof. Background Art

[0002] Panax notoginseng, as a precious medicinal plant of the genus Panax in the Araliaceae family, has a long history of medicinal use and a wide range of medicinal values. Its roots and rhizomes contain a variety of bioactive ingredients, such as saponins, flavonoids and polysaccharides, which are widely used in the prevention and treatment of cardiovascular and cerebrovascular diseases. With the growing global demand for natural medicines and health products, the market demand for Panax notoginseng is also continuing to expand, especially in the Asian market, where Panax notoginseng has become an important cash crop.

[0003] However, the high economic value of Panax notoginseng has also led to the proliferation of counterfeit and inferior products in the market. For example, the wrong substitution and mixing of Panax notoginseng powder, ginseng powder and American ginseng powder, adulteration of Panax notoginseng powder, and the substitution and mixing of Panax notoginseng flower with American ginseng flower and ginseng flower. These counterfeit and inferior products not only harm the interests of consumers, but also pose a potential threat to public health. Therefore, ensuring the authenticity and purity of Panax notoginseng and its products is crucial to maintaining market order and consumer rights.

[0004] Although traditional methods for identifying Panax notoginseng, such as microscopic observation, thin-layer chromatography, and chemical analysis, can identify Panax notoginseng to a certain extent, these methods are often time-consuming, require professional equipment and technicians, and are difficult to meet the needs of large-scale rapid detection. In addition, with the development of molecular biology techniques, DNA-based molecular identification methods have been valued for their accuracy and repeatability. However, existing molecular detection methods, such as PCR amplification, DNA sequencing and bioinformatics analysis, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), etc., are usually complex to operate, costly, and have a long detection cycle, which limits their widespread promotion in practical applications. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention innovatively introduces the Ladder-shape melting temperature is othermal amplification (LMTIA) technology (for technical principles, see Wang Deguo, et al. Ladder-shape melting temperature is othermal amplification of nucleic acids. Biotechniques, 2021, 71 (1): 358-369, or patent CN2020111054053 or PCT / CN2020 / 133584), designs LMTIA primers, and combines Proofman probes to develop a rapid, sensitive and specific method for detecting the components of Panax notoginseng. This method not only provides an efficient means of authenticity identification for Panax notoginseng seeds, seedlings, raw medicinal materials and their derivatives, but also greatly simplifies the detection process, reduces costs and shortens detection time.

[0006] The present invention is achieved through the following technical solutions:

[0007] According to a first aspect of an embodiment of the present invention, the present invention provides a LMTIA primer set and a Proofman probe specific for the ITS2 sequence of Panax notoginseng; the sequences of the primer set and the probe are as follows:

[0008] Pn-F1: 5'-TGGGCCAACCGCGCttttCCCCAACCCATCATTCCCTC-3'

[0009] Pn-B1: 5'-TGCGAGTCCTTGGCGAtttACAACCACCACTTGTCGTGA-3'

[0010] Pn-LF: 5'-GAGACACGGGGAGGCCATTAT-3'

[0011] PnProbe:5'-BHQ1-CCCTCCGCC-3'-6-FAM.

[0012] According to the second aspect of an embodiment of the present invention, the present invention provides a kit for authenticity detection of Panax notoginseng and its derivative products (foods, medicines, health products, etc. containing Panax notoginseng ingredients), so that the detection of Panax notoginseng ingredients is more convenient and standardized; the kit comprises the primer set and probe described in the first aspect.

[0013] According to the third aspect of an embodiment of the present invention, the present invention provides an application of the LMTIA primer set and Proofman probe according to the first aspect or the kit according to the second aspect in the detection of Panax notoginseng seeds, seedlings, Panax notoginseng raw materials and their derivative products (foods, medicines, health products and other products containing Panax notoginseng ingredients).

[0014] According to the fourth aspect of an embodiment of the present invention, the present invention provides a rapid detection method for Panax notoginseng and its derivatives based on Proofman-LMTIA, comprising the following steps: taking a sample to be tested, extracting genomic DNA of the sample, and preparing a Proofman-LMTIA reaction system using the LMTIA primer set and Proofman probe described in the first aspect or the kit described in the second aspect; placing the prepared Proofman-LMTIA reaction system in an isothermal device for isothermal amplification to detect whether Panax notoginseng components are present in the sample.

[0015] As a further preferred technical solution, the method for judging whether Panax notoginseng components are present in the test sample is specifically as follows: in the sample test tube after the reaction is completed, if a fluorescent signal can be detected in the green fluorescent channel (FAM) and an exponential amplification curve appears in the amplification result graph, the sample contains Panax notoginseng components; if no fluorescent signal can be detected in the green fluorescent channel (FAM) and there is no exponential amplification curve in the amplification result graph, the sample does not contain Panax notoginseng components.

[0016] As a further preferred technical solution, the isothermal equipment includes a real-time fluorescence quantitative PCR system.

[0017] As a further preferred technical solution, the molar ratio of Pn-F1:Pn-B1:Pn-LF:PnProbe in the Proofman-LMTIA reaction system is 4:4:1:7.5.

[0018] As a further preferred technical solution, the Proofman-LMTIA reaction system is 10 μL, and its components are: 5 μL of 2×Mix premix, 0.4 μL of GPV8 ultra-fidelity DNA polymerase, 0.16 μL of primer Pn-F1, 0.16 μL of Pn-B1, 0.04 μL of primer Pn-LF, 0.3 μL of PnProbe, 2 μL of genomic DNA template, and 1.94 μL of DEPC-treated water.

[0019] As a further preferred technical solution, the temperature of the isothermal amplification is 65° C., the number of cycles is 30-60, a fluorescence signal is collected every 45 seconds, and a total of 30-60 fluorescence signals are collected.

[0020] The present invention proposes a rapid detection method for Panax notoginseng components based on Proofman-LMTIA, which realizes rapid, accurate and low-cost detection of Panax notoginseng components. The method of the present invention realizes high-specificity and high-sensitivity detection of Panax notoginseng components by designing LMTIA primers and Proofman probes. Compared with traditional detection technologies, the present invention has the advantages of simplified operation process, low-cost equipment requirements, and rapid detection (only about 30 minutes), and has a wide range of applications. The method of the present invention is not only suitable for the detection of raw materials such as Panax notoginseng seeds, seedlings, roots and rhizomes, and medicinal pieces, but can also be used for the authenticity identification of terminal products such as food, medicine, and health products containing Panax notoginseng components.

[0021] The beneficial effects of the present invention include:

[0022] The Proofman-LMTIA primer set and probe for detecting notoginseng components of the present invention have good specificity and sensitivity, fast detection speed, and can achieve accurate detection of notoginseng components in samples under constant temperature conditions.

[0023] The LMTIA detection method for detecting Panax notoginseng components of the present invention is simple and quick, and can achieve rapid and accurate detection of Panax notoginseng components in a sample by amplifying for about 30 minutes under isothermal conditions, and has good repeatability and high stability.

[0024] The LMTIA primer set and Proofman probe of the present invention have higher amplification efficiency and best specificity at 65°C.

[0025] The absolute sensitivity of the LMTIA primer set and Proofman probe of the present invention can reach 10 pg / μL.

[0026] The LMTIA primer set and Proofman probe of the present invention can not only be used to detect raw materials such as Panax notoginseng seeds, seedlings, roots and rhizomes, and medicinal pieces, but can also be used to authenticate the authenticity of terminal products such as foods, medicines, health products, and beverages containing Panax notoginseng ingredients.

[0027] The present invention not only solves the deficiencies of the prior art in terms of operational complexity, cost and detection time, but also provides an effective solution for the rapid and accurate identification of Panax notoginseng and its derivatives, and has important market application prospects and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The SYBR Green I-LMTIA test results of the Panax notoginseng primers of the embodiment of the present invention;

[0030] Figure 2 This is the Proofman-LMTIA temperature optimization test result of Panax notoginseng according to the embodiment of the present invention;

[0031] Figure 3 This is the Proofman-LMTIA specificity test result of Panax notoginseng according to the embodiment of the present invention;

[0032] Figure 4 This is the Proofman-LMTIA sensitivity test result of Panax notoginseng according to the embodiment of the present invention;

[0033] Figure 5 This is the Proofman-LMTIA detection limit test result of the simulated adulteration of Panax notoginseng and ginseng according to the embodiment of the present invention;

[0034] Figure 6 This is the Proofman-LMTIA detection limit test result of the simulated adulteration of Panax notoginseng-American ginseng according to the embodiment of the present invention;

[0035] Figure 7 The Proofman-LMTIA test results of the Panax notoginseng decoction pieces according to the embodiment of the present invention;

[0036] Figure 8 The Proofman-LMTIA test results of the medicine containing Panax notoginseng ingredients in the embodiment of the present invention;

[0037] Fig. 9 The amplification result of the ITS2 sequence of Panax notoginseng in the embodiment of the present invention; M: DNA molecular weight standard; 1, 2: Panax notoginseng;

[0038] Fig.10 The results of restriction enzyme digestion detection of the Panax notoginseng pMD19T-PnITS2 plasmid of the embodiment of the present invention; M: DNA molecular weight standard; 1-7: pMD19T-PnITS2 plasmids of different clones. DETAILED DESCRIPTION

[0039] 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.

[0040] Unless otherwise defined, all professional terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention; unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0041] Based on the sequence characteristics of the internal transcribed spacer 2 (ITS2), the present invention uses the DNA barcode molecular identification method to develop an innovative rapid detection technology for the Chinese medicinal material Panax notoginseng and its derivatives. There are multiple copies of the ITS2 sequence in the plant genome, which can cope with the problem of partial degradation of genomic DNA that occurs during the processing of medicinal materials. The present invention selects the ITS2 sequence as the detection target, and through carefully designed primers and probes, combined with the Proofman-LMTIA technology, it realizes efficient authenticity identification of Panax notoginseng and its derivatives.

[0042] Example 1

[0043] Design and screening of primers for Panax notoginseng

[0044] Reagents and instruments: The reagents are commonly used molecular biology experimental reagents, and the instruments are standard equipment in molecular biology laboratories. All primers were synthesized by Shanghai Jierui Bioengineering Co., Ltd. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional molecular biology methods.

[0045] (1) Design of primers and probes

[0046] DNMAN 9 software was used to compare and analyze the ITS2 sequences of Panax notoginseng, American ginseng and ginseng, and the difference regions between Panax notoginseng, American ginseng and ginseng were accurately identified. Subsequently, Oligo 7 software was used to find the region with a trapezoidal or semi-trapezoidal melting temperature Tm for the characteristic difference sequences in the ITS2 sequence of Panax notoginseng. According to the LMTIA primer design principle, the online PCR primer design software Primer 3PLUS (http: / / www.primer3plus.com) was used to design LMTIA primers and Proofman probes. After multiple rounds of screening and optimization, the following primer set was obtained. The specific sequence is as follows:

[0047] Pn-F1: 5'-TGGGCCAACCGCGCttttCCCCAACCCATCATTCCCTC-3'

[0048] Pn-B1: 5'-TGCGAGTCCTTGGCGAttttACAACCACCACTTGTCGTGA-3'

[0049] Pn-LF: 5'-GAGACACGGGGAGGCCATTAT-3'

[0050] PnProbe:5'-BHQ1-CCCTCCGCC-3'-6-FAM.

[0051] (2) Extraction and detection of genomic DNA from Panax notoginseng, American ginseng and Panax ginseng

[0052] Take 5g of dried medicinal materials and grind them into powder using a Chinese medicinal material grinder. Weigh 100mg of sample and use a high-efficiency plant genomic DNA extraction kit (DP350, Tiangen Biochemical Technology Beijing Co., Ltd.) to extract genomic DNA. After extraction, use an ultra-micro nucleic acid protein analyzer (Nanodrop One, Thermo Fisher Scientific, USA) to measure the concentration and purity of genomic DNA. 260 / A 280 The 1.6-2.0 can be used for the next step of detection. The extracted genomic DNA is stored in a -20℃ refrigerator for future use.

[0053] (3) Detection of Panax notoginseng primers

[0054] Primer detection was performed using the SYBR Green I fluorescent dye method to determine the availability of primers. In a clean bench, the following reagents were added to a 100 μL PCR tube: 5 μL of Mix premix (in the embodiment of the present invention, the premix contains Bst polymerase, dNTP, 2×Bst polymerase buffer), 0.13 μL of Pn-F1 (10 μmol / L), 0.13 μL of Pn-B1 (10 μmol / L), 0.065 μL of Pn-LF (10 μmol / L), 2 μL of genomic DNA (10 ng / μL), and 2.675 μL of ddH2O. The Archimed X4 medical fluorescence quantitative PCR instrument (Kunpeng Gene Beijing Scientific Instrument Co., Ltd.) was used for detection, and the reaction temperature was set to 52°C, 54°C, 56°C, and 58°C. The number of cycles was set to 40, and the fluorescence signal was collected every 90 seconds, and a total of 40 fluorescence signals were collected. The experimental group was Panax notoginseng genomic DNA (10 ng / μL), and the negative control was DEPC-treated water. The results were analyzed using ArchimedAnalyzer software (Kunpeng Gene Beijing Scientific Instrument Co., Ltd.).

[0055] In the embodiment of the present invention, if the fluorescent signal can be detected in the green fluorescence (SYBR Green I) channel, and an exponential amplification curve appears in the amplification result graph, it indicates that the genomic DNA of the extracted sample has been successfully amplified; if the fluorescent signal cannot be detected in the green fluorescence (SYBR Green I) channel, and there is no exponential amplification curve in the amplification result graph, it indicates that the genomic DNA of the extracted sample has failed to be amplified. The criterion for primer detection is whether the sample genomic DNA has been successfully amplified. It is explained here that in the embodiment of the present invention, a real-time fluorescence quantitative PCR system is used to perform an isothermal amplification reaction. In other embodiments, other isothermal devices can also be used.

[0056] Test results such as Figure 1 As shown in the figure, the curve marked with temperature corresponds to Panax notoginseng genomic DNA, and the one marked with DEPC-treated water is a negative control. Observing the amplification curve, it can be seen that the negative control has no amplification. The F1 primer set at 58°C, 56°C, 54°C, and 52°C, the Panax notoginseng genomic DNA began to show amplification curves from the 12th, 11th, 13th, and 15th cycles, respectively, and the number of cycles at 56°C was the smallest, so the F1 primer set can be used for subsequent experiments.

[0057] Example 2

[0058] Proofman-LMTIA temperature optimization test of Panax notoginseng

[0059] Temperature optimization of the Proofman-LMTIA reaction system. In a clean bench, the following reaction system was added to a 100 μL PCR tube: 5 μL of 2×Mix premix (in the embodiment of the present invention, the premix contains Bst polymerase, dNTP, 2×Bst polymerase buffer), 0.4 μL of GPV8 ultra-fidelity DNA polymerase (2U / μL, Anhui Global Gene Technology Co., Ltd.), 0.16 μL of primer Pn-F1 (10 μmol / L), 0.16 μL of Pn-B1 (10 μmol / L), 0.04 μL of primer Pn-LF (10 μmol / L), 0.3 μL of Proofman probe PnProbe (10 μmol / L), 2 μL of Panax notoginseng genomic DNA template, and 1.94 μL of DEPC-treated water. The Gentier 96E fully automatic medical PCR analysis system (Xi'an Tianlong Technology Co., Ltd.) was used for detection. The reaction temperatures were 59°C, 61°C, 63°C, and 65°C, respectively. The number of cycles was set to 40, and the fluorescence signal was collected every 45 seconds, for a total of 40 fluorescence signals. The test sample was Panax notoginseng genomic DNA, and the negative control was DEPC-treated water. Two parallel samples were set for each experiment, and the test results were statistically and observed using the Gentier 96E fully automatic medical PCR analysis system software.

[0060] According to an exemplary embodiment of the present invention, in a sample test tube that has completed the reaction, if a fluorescent signal can be detected in the green fluorescent channel (FAM) and an exponential amplification curve appears in the amplification result graph, it can be determined that the sample contains Panax notoginseng; if no fluorescent signal can be detected in the green fluorescent channel (FAM) and there is no exponential amplification curve in the amplification result graph, it can be determined that the sample does not contain Panax notoginseng. The following embodiments will not be repeated.

[0061] Temperature optimization test results are as follows Figure 2 As shown in the figure, the curve marked with temperature corresponds to Panax notoginseng genomic DNA, and DEPC-treated water is a negative control. When the reaction temperature is 65℃, 63℃, 61℃, and 59℃, Panax notoginseng genomic DNA starts amplification from the 16th, 16th, 20th, and 27th cycles, respectively. The number of amplification cycles is the smallest when the reaction temperature is 65℃ and 63℃. Since the higher the temperature in nucleic acid amplification, the stronger the specificity of amplification, 65℃ is selected as the optimal reaction temperature for Panax notoginseng Proofman-LMTIA reaction.

[0062] Example 3

[0063] Proofman-LMTIA specificity test of Panax notoginseng

[0064] 10 ng / μL of Panax notoginseng genomic DNA was used as a positive control, 10 ng / μL of ginseng genomic DNA, 10 ng / μL of American ginseng genomic DNA and DEPC-treated water were used as negative controls, and the optimized Proofman-LMTIA reaction system established in Example 2 was used. The reaction was carried out using the Gentier 96E fully automatic medical PCR analysis system, the temperature was set to 65°C, 40 cycles were performed, and the fluorescence signal was collected every 45s, for a total of 40 fluorescence signals. Two parallel samples were set for each reaction, and the test results were statistically and visualized using the Gentier 96E fully automatic medical PCR analysis system.

[0065] Specific test results such as Figure 3 As shown in the figure, the exponential amplification curve corresponds to Panax notoginseng genomic DNA, and the baseline curve corresponds to American ginseng genomic DNA, Panax ginseng genomic DNA and DEPC treated water; Panax notoginseng genomic DNA has good amplification efficiency, while American ginseng genomic DNA, Panax ginseng genomic DNA and negative control have no amplification, indicating that the Proofman-LMTIA reaction system established in the present invention has good specificity for American ginseng genomic DNA and Panax ginseng genomic DNA.

[0066] Example 4

[0067] Proofman-LMTIA sensitivity test of Panax notoginseng

[0068] The extracted Panax notoginseng genomic DNA was diluted to 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 0.1 pg / μL, respectively, and DEPC-treated water was used as a negative control. The DNA was added to the optimized Proofman-LMTIA reaction system (see Example 2 for details). Two parallel samples were set for each reaction, the temperature was set to 65°C, and the fluorescence signal was collected every 45 s. A total of 40 fluorescence signals were collected, and each group of experiments was repeated three times to determine the best reaction result.

[0069] Sensitivity test results are as follows Figure 4 As shown, 1ng / μL, 100pg / μL, and 10pg / μL of Panax notoginseng genomic DNA were well amplified, while 1pg / μL, 0.1pg / μL of Panax notoginseng genomic DNA and DEPC-treated water were not amplified. Therefore, the sensitivity of this method is 10pg / μL.

[0070] Example 5

[0071] Proofman-LMTIA detection limit test of Panax notoginseng

[0072] The extracted Panax notoginseng genomic DNA was mixed with ginseng genomic DNA, and the Panax notoginseng genomic DNA was mixed with ginseng genomic DNA at 0%, 0.1%, 1.0%, 5.0%, 10%, 20% and 100% respectively. These mixed samples were added to the optimized Proofman-LMTIA reaction system (see Example 2 for details). The Gentier 96E fully automatic medical PCR analysis system was used for detection, and DEPC-treated water was used as a negative control. Each group of experiments was repeated twice. The temperature was set at 65°C, and the fluorescence signal was collected every 45s, and a total of 40 fluorescence signals were collected.

[0073] The detection limit test results are as follows Figure 5 As shown in the figure, the curves marked by 100%, 20%, 10%, 5%, 1% and 0.1% correspond to samples with a volume fraction of 100%, 20%, 10%, 5.0%, 1.0% and 0.1% of Panax notoginseng genomic DNA, respectively, and the baseline corresponds to a sample with a volume fraction of 0% of Panax notoginseng genomic DNA and negative control DEPC treated water; observing the amplification curve, it can be seen that the prepared Panax notoginseng genomic DNA was successfully amplified in mixed samples of 100%, 20%, 10%, 5%, 1% and 0.1%, while no amplification occurred in the mixed sample of 0% and the negative control. Therefore, the detection limit of this method for ginseng adulteration is 0.1%.

[0074] Similarly, the extracted Panax notoginseng genomic DNA was mixed with American ginseng genomic DNA, and the Panax notoginseng genomic DNA was mixed with American ginseng genomic DNA at 0%, 0.1%, 1.0%, 5.0%, 10%, 20% and 100% respectively. These mixed samples were added to the optimized Proofman-LMTIA reaction system (see Example 2 for details). The Gentier 96E fully automatic medical PCR analysis system was used for detection, and DEPC-treated water was used as a negative control. Each group of experiments was repeated twice. The temperature was set at 65°C, and the fluorescence signal was collected every 45s, and a total of 40 fluorescence signals were collected.

[0075] The detection limit test results are as follows Figure 6 As shown in the figure, the curves marked by 100%, 20%, 10%, 5%, and 1% correspond to samples with a volume fraction of 100%, 20%, 10%, 5.0%, and 1.0% of the genome DNA of Panax notoginseng, respectively, and the baseline corresponds to samples with a volume fraction of 0.1% and 0% of the genome DNA of Panax notoginseng and negative control DEPC treated water; observing the amplification curve, it can be seen that the prepared genome DNA of Panax notoginseng was successfully amplified in mixed samples of 100%, 20%, 10%, 5%, and 1%, while no amplification occurred in mixed samples of 0.1% and 0% and negative control. Therefore, the detection limit of this method for adulteration of American ginseng is 1%.

[0076] Example 6

[0077] Proofman-LMTIA testing of Panax notoginseng decoction products

[0078] Take 7 portions of Panax notoginseng decoction pieces from the market, extract genomic DNA according to the method of Example 1, and detect according to the method of Example 2. Use Gentier 96E fully automatic medical PCR analysis system for reaction, set the temperature to 65°C, 60 cycles, collect fluorescence signals every 45s, collect 60 fluorescence signals in total, and set two parallel samples for each reaction. Use Gentier96E fully automatic medical PCR analysis system to count and visualize the test results. The results are as follows Figure 7 As shown, 7 samples of Panax notoginseng genomic DNA were successfully amplified, and the negative control DEPC-treated water did not produce amplification, indicating that this method can be used for the rapid detection of Panax notoginseng decoction products.

[0079] Example 7

[0080] Proofman-LMTIA testing of drugs containing Panax notoginseng

[0081] Take 7 commercially available medicines containing Panax notoginseng ingredients and 1 Panax notoginseng slices, extract genomic DNA according to the method of Example 1, and detect according to the method of Example 2. Use Gentier 96E fully automatic medical PCR analysis system for reaction, set the temperature to 65°C, 60 cycles, collect fluorescence signals every 45s, and collect 60 fluorescence signals in total. Set 3 parallel samples for each reaction, and use Gentier 96E fully automatic medical PCR analysis system to count and visualize the test results. Use Panax notoginseng slices genomic DNA as positive control and DEPC treated water as negative control. The test results are as follows: Figure 8 As shown in the figure, the genomic DNA of the positive control, Xinkeshu capsule, Dieda pill, Danqi tablet, Huoxuezhitong tablet, Wenxin granule, Fufang Danshen dripping pill, and Sanqi Shangyao tablet all produced good amplification, while the negative control DEPC-treated water had no amplification, indicating that this method can be used for the rapid detection of drugs containing Panax notoginseng.

[0082] Example 8

[0083] Preparation of Panax notoginseng pMD19T-PnITS2 plasmid

[0084] In a clean bench, add the following reagents to a 200 μL PCR tube: 25 μL Max DNA Polymerase (2×) premix (in the embodiment of the present invention, the premix contains high-fidelity DNA polymerase, dNTP, 2× polymerase buffer), 2 μL of ITS2-F (10 μmol / L), 2 μL of ITS2-R (10 μmol / L), 4 μL of genomic DNA, and 17 μL of ddH2O. The reaction conditions are: (98°C 10s, 53°C 5s, 72°C 10s) × 30 cycles. After the PCR reaction is completed, an A-tailing reaction is performed. The PCR products are separated by agarose gel electrophoresis ( Fig. 9 ), after the electrophoresis, gel cutting, gel recovery and ligation to pMD19T vector were performed. E. coli DH5α was transformed by heat shock method, and then coated on AIX plate, cultured, picked, shaken and plasmid was extracted. Then, enzyme digestion was performed to detect whether the target fragment had been successfully inserted into the vector ( Fig.10 ). Finally, it was sent to Shanghai Biotech Co., Ltd. for sequencing. After sequencing, the pMD19T-PnITS2 plasmid of Panax notoginseng was obtained. This plasmid was used as the positive control of the Proofman-LMTIA method in the kit.

[0085] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The LMTIA primer set and Proofman probe for detecting Panax notoginseng are characterized by: The primer set and probe use Panax notoginseng ITS2 as the target sequence, wherein the sequences of the primer set and probe are: Pn-F1: 5'-TGGGCCAACCGCGCttttCCCCAACCCATCATTCCCTC-3' Pn-B1: 5'-TGCGAGTCCTTGGCGAttttACAACCACCACTTGTCGTGA-3' Pn-LF: 5'-GAGACACGGGGAGGCCATTAT-3' PnProbe:5'-BHQ1-CCCTCCGCC-3'-6-FAM.

2. A kit for detecting the authenticity of Panax notoginseng, characterized in that: The kit comprises the primer set and probe according to claim 1.

3. Use of the LMTIA primer set and Proofman probe described in claim 1 or the kit described in claim 2 in the detection of Panax notoginseng seeds, seedlings, Panax notoginseng raw materials, foods containing Panax notoginseng ingredients, medicines or health products.

4. A rapid detection method of Panax notoginseng based on Proofman-LMTIA, characterized in that: include: Take the sample to be tested, extract the sample genomic DNA, and prepare the Proofman-LMTIA reaction system using the LMTIA primer set and Proofman probe described in claim 1 or the kit described in claim 2; place the prepared Proofman-LMTIA reaction system in an isothermal device for isothermal amplification to detect whether Panax notoginseng components are present in the sample.

5. The rapid detection method of Panax notoginseng based on Proofman-LMTIA according to claim 4, characterized in that: The method for judging whether the detection sample contains Panax notoginseng is as follows: in the sample detection tube after the reaction is completed, if a green fluorescence signal can be detected in the green fluorescence channel and an exponential amplification curve appears in the amplification result graph, the sample contains Panax notoginseng; If no green fluorescence signal is detected in the green fluorescence channel and there is no exponential amplification curve in the amplification result graph, the sample does not contain Panax notoginseng components.

6. The rapid detection method of Panax notoginseng based on Proofman-LMTIA according to claim 4, characterized in that: The isothermal equipment includes a real-time fluorescence quantitative PCR system.

7. The rapid detection method of Panax notoginseng based on Proofman-LMTIA according to claim 4, characterized in that: The molar ratio of Pn-F1:Pn-B1:Pn-LF:PnProbe in the Proofman-LMTIA reaction system is 4:4:1:7.

5.

8. The rapid detection method of Panax notoginseng based on Proofman-LMTIA according to claim 7, characterized in that: The Proofman-LMTIA reaction system is 10 μL, and its components are: 5 μL of 2×Mix premix, 0.4 μL of GPV8 ultra-fidelity DNA polymerase, 0.16 μL of primer Pn-F1, 0.16 μL of Pn-B1, 0.04 μL of primer Pn-LF, 0.3 μL of PnProbe, 2 μL of genomic DNA template, and 1.94 μL of DEPC-treated water.

9. The rapid detection method of Panax notoginseng based on Proofman-LMTIA according to any one of claims 4 to 8, characterized in that: The temperature of the isothermal amplification is 65° C., the number of cycles is 40-60, and the fluorescence signal is collected every 45 seconds, and a total of 40-60 fluorescence signals are collected.

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