Specific primer and method for rapidly identifying Chinese herbal medicine semen allii tuberosi and semen allii fistulosi

Abstract

The invention discloses a specific primer and a method for rapidly identifying Chinese herbal medicine semen allii tuberosi and semen allii fistulosi. The specific primer comprises the following developed specific primer pairs: JP1-F and JP1-R; according to the present invention, the specific sequences of the semen allii tuberosi and the semen allii fistulosi can be amplified by using the fluorescent quantitative PCR instrument, the nucleic acid dyeing electrophoresis is not required, the rapid identification can be achieved by detecting the fluorescence signal, the whole identification period only needs 3 h, and the method has characteristics of simple operation, strong specificity, no toxic reagent, no influence of the sample appearance, the production place and other factors, and can be used for the rapid identification of the semen allii tuberosi and the semen allii fistulosi. And the method has a relatively strong application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of molecular identification of traditional Chinese medicines, and specifically proposes a specific primer and method for rapidly identifying the traditional Chinese medicines leek seeds and onion seeds. Background Art

[0002] Both leek seeds and onion seeds are dried mature seeds of the Allium genus of the Liliaceae family. They are varieties with clear medicinal value in my country's traditional Chinese medicinal materials system. There are significant differences in medicinal efficacy and clinical applications between the two. Accurate identification is crucial to ensuring the safety and efficacy of medication.

[0003] Among them, leek seeds are the dried mature seeds of Allium tuberosum Rottl. ex Spreng., a plant of the Liliaceae family. They are star-shaped, semi-circular or semi-oval, slightly flat, 2~4mm long, 1.5~3mm wide, black in surface, one side is raised and rough with fine reticular wrinkles, the other side is slightly concave with inconspicuous wrinkles, the top is blunt, the base is slightly pointed with a dotted hilum, the texture is hard, the smell is peculiar and the taste is slightly spicy; in terms of medicinal effects, leek seeds have the core functions of warming and nourishing the liver and kidneys, strengthening yang and consolidating essence, and are mainly used clinically to treat symptoms such as low back and knee pain, impotence and spermatorrhea, enuresis and frequent urination, and leucorrhea caused by liver and kidney deficiency. It is one of the commonly used Chinese medicinal materials in men's and gynecology.

[0004] Scallion seeds are the dried mature seeds of Allium jistulosum L., a plant of the Liliaceae family. They are triangular and flat-oval in shape, with one side slightly concave and the other side raised with 1 to 2 ridges. They are 3 to 4 mm long and 2 to 3 mm wide. The surface is black, mostly smooth or occasionally with sparse wrinkles, and the concave surface is smooth. Its medicinal effects are concentrated on warming the kidney, improving eyesight, and detoxifying. Its clinical application direction is clearly different from that of leek seeds, and it cannot be used as a substitute for leek seeds.

[0005] However, since leek seeds and onion seeds come from the same closely related species and the morphological characteristics of the seeds after drying (such as color, size, and surface texture) are highly similar, traditional identification methods rely on manual observation of morphological characteristics and are highly subjective. Different appraisers have different judgment criteria for characteristics such as the "density of network wrinkles" and the "degree of ridgeline prominence," which can easily lead to misjudgment. At the same time, when samples are damaged in appearance such as breakage, mildew, or insect bites, or when there are slight variations in morphology due to differences in origin or growth environment, the accuracy of traditional morphological identification methods is further reduced, and they cannot meet the precision requirements of quality control of Chinese medicinal materials.

[0006] To solve the limitations of morphological identification, molecular biology-based identification methods have appeared in the prior art. For example, patent CN108517370A discloses a technology for identifying by restriction enzyme Rsa#I enzyme digestion of Allium macranthum and Allium chinense ribosomal DNA internal transcribed spacer (ITS) sequences, and then observing the size of the enzyme digestion fragments by agarose gel electrophoresis. However, this method still has significant defects: first, the entire identification process includes four core steps of "nucleic acid extraction, PCR amplification, enzyme digestion reaction, and electrophoresis detection", which has many operation links, resulting in an identification period of 8-10 hours, which cannot meet the rapid detection requirements; second, toxic dyes such as agarose gel and EB (ethidium bromide) are required in the electrophoresis process, and EB has strong carcinogenicity, which not only causes biological pollution to the laboratory environment, but also poses a threat to the occupational health of the operators; third, when the PCR product concentration is low, the electrophoresis cannot clearly display the enzyme digestion fragment band, resulting in inaccurate determination of the sample type, especially when detecting trace adulteration samples, this defect is more prominent.

[0007] At present, the confusion and adulteration of Allium macranthum and Allium chinense frequently occur in the market of traditional Chinese medicinal materials, and the existing identification technologies either have strong subjectivity and low accuracy or have long process, large pollution, and insufficient sensitivity, which are difficult to meet the actual needs of rapid screening by drug regulatory departments and batch quality control by enterprises. Therefore, developing a technical solution for quickly and accurately identifying Allium macranthum and Allium chinense with simple operation, strong specificity, no toxic reagents, and no influence of sample appearance and origin has become a key problem to be solved in the field of molecular identification of traditional Chinese medicinal materials, and has important practical significance for standardizing the order of the market of traditional Chinese medicinal materials and ensuring the safety of clinical medication. SUMMARY

[0008] Therefore, the core purpose of the present application is to provide a specific primer and method for quickly identifying traditional Chinese medicinal materials Allium macranthum and Allium chinense, which realizes the goal of "no enzyme digestion and electrophoresis, no toxic reagents, and identification completed within 3 hours" by designing specific primers and optimizing the identification technology path, while ensuring accurate determination of single varieties and mixed adulteration samples.

[0009] The technical solution of the present application is realized as follows: the present application provides a specific primer for quickly identifying traditional Chinese medicinal materials Allium macranthum and Allium chinense. Through deep alignment analysis of the internal transcribed spacer 2 (ITS2) sequences of Allium macranthum and Allium chinense, it is found that there are three highly conserved differential base regions (which are specific to Allium macranthum or Allium chinense and are not homologous to other related species) in the ITS2 sequences. Based on the above differential base regions, two sets of specific primer pairs for Allium macranthum and Allium chinense are designed, as follows: 1. Allium macranthum specific primer pair (JP1-F / JP1-R): Upstream primer JP1-F: the nucleotide sequence is shown in SEQ ID No: 1 in the sequence listing, specifically 5'-ACGGTAAACTTACACTGTGAG-3'; Downstream primer JP1-R: the nucleotide sequence is shown in SEQ ID No: 2 in the sequence listing, specifically 5'-GTGTTACATCGTGCGTAGGA-3'.

[0010] The primer pair can only specifically bind to the ITS2 specific sequence in the genome of Allium macrostemon Bunge and initiate amplification, and cannot bind to the nucleic acid sequence of Allium chinense or other species.

[0011] 2. Allium chinense specific primer pair (JP2-F / JP2-R): Upstream primer JP2-F: the nucleotide sequence is shown in SEQ ID No: 3 in the sequence listing, specifically 5'-CGTTGTTTGCACTATTTGCAAGA-3'; Downstream primer JP2-R: the nucleotide sequence is shown in SEQ ID No: 4 in the sequence listing, specifically 5'-TGACGCTTTCCCCAGACTAC-3'.

[0012] The primer pair can only specifically bind to the ITS2 specific sequence in the genome of Allium chinense and initiate amplification, and cannot bind to the nucleic acid sequence of Allium macrostemon Bunge or other species.

[0013] The application also provides a rapid identification method, based on the above specific primers, the rapid identification method provided by the application comprises the following steps: 1. Extraction of total nucleic acid of sample: using a plant genomic DNA extraction kit, such as a special nucleic acid extraction kit suitable for traditional Chinese medicinal materials, to extract total nucleic acid of the sample to be tested; before extraction, the sample to be tested, such as Allium macrostemon Bunge, Allium chinense or a suspected mixed sample, can be ground into powder to improve the nucleic acid extraction efficiency; during the extraction process, no toxic lysis reagent is needed, and high-quality total nucleic acid can be obtained through conventional kit operation.

[0014] 2. Fluorescent PCR amplification: using total nucleic acid of the sample to be tested as a template, and using the above two sets of specific primer pairs (JP1-F / JP1-R, JP2-F / JP2-R) for fluorescent PCR amplification; Fluorescent PCR reaction system (total volume 20 μL): 50 ng DNA template, 10 μL SYBR qPCR Master Mix (containing fluorescent dye, which can monitor the amplification process in real time), 1 μL upstream primer (JP1-F or JP2-F), 1 μL downstream primer (JP1-R or JP2-R), and the rest is supplemented with sterile double distilled water to 20 μL; The fluorescent PCR amplification procedure is as follows: 95 DEG C pre-denaturation for 2 min to completely denature the template DNA, and then 40 cycles of 95 DEG C denaturation for 5 s to unwind the DNA double strand, 60 DEG C annealing and extension for 30 s to enable the primer to specifically bind to the template and complete the fragment extension; the procedure can be automatically operated by a conventional fluorescent quantitative PCR instrument without manual intervention.

[0015] 3. Result reading and determination: the fluorescent signals of each primer pair are monitored and read in real time by the fluorescent quantitative PCR instrument, and the sample type is determined according to the presence or absence of the fluorescent signals and the corresponding primer pair: If only the JP1-F / JP1-R primer pair has obvious fluorescent amplification curve, it is determined that the sample to be tested is the semen Allii victorialis; If only the JP2-F / JP2-R primer pair has obvious fluorescent amplification curve, it is determined that the sample to be tested is the semen Allii fistulosi; If the JP1-F / JP1-R and JP2-F / JP2-R primer pairs all have obvious fluorescent amplification curve, it is determined that the sample to be tested is a mixture of the semen Allii victorialis and the semen Allii fistulosi.

[0016] Based on the specific primers and the fluorescent PCR identification method, the application further provides a kit for rapidly identifying the semen Allii victorialis and the semen Allii fistulosi, which at least comprises: 1. The two sets of specific primer pairs (JP1-F / JP1-R, JP2-F / JP2-R) described above, the primers are purified and can be directly used for fluorescent PCR reaction; 2. The basic reagents required for fluorescent PCR reaction, including SYBR qPCR Master Mix (containing DNA polymerase, dNTPs, fluorescent dye, etc.), RNase-free double distilled water (to avoid RNA pollution affecting the amplification result); 3. Optional auxiliary reagents or tools, such as lysis solution and washing solution required for nucleic acid extraction, or sterile grinding beads for sample grinding, to simplify the sample pretreatment operation and improve the practicability of the kit.

[0017] The application has the following beneficial effects relative to the prior art: The specific primer pairs of the application are designed based on the specific difference region of the ITS2 sequence of the semen Allii victorialis and the semen Allii fistulosi, and can only bind to and amplify the nucleic acid sequence of the target species, without reacting with other related species or impurity nucleic acids; at the same time, the identification is directly determined by the fluorescent signal, avoiding the subjectivity of morphological identification and the fragment misjudgment problem of enzyme digestion electrophoresis, and even if the semen Allii victorialis and the semen Allii fistulosi are mixed at a very low ratio of 1:19, the two components can still be accurately detected, and the identification accuracy can reach 100%.

[0018] The whole identification process only needs 3 hours (about 1 hour for nucleic acid extraction, about 1.5 hours for fluorescent PCR amplification, and about 0.5 hours for result reading), which is more than 60% higher in efficiency compared with the existing enzyme digestion electrophoresis method, and can meet the timeliness requirements of on-site rapid screening of drug regulatory departments and batch sample detection of enterprises.

[0019] Without the steps of restriction endonuclease digestion and agarose gel electrophoresis, the use of toxic reagents such as EB is avoided, and the biological pollution to the laboratory environment and the health risk of the operator are fundamentally eliminated; at the same time, the fluorescent PCR reaction can be completed automatically by the instrument without complex manual operation, which reduces the requirement for the professional skills of the operator and facilitates the popularization and application of the primary laboratory.

[0020] The identification process is based on nucleic acid sequence and is not affected by factors such as sample appearance, origin, and growth environment, and can accurately identify intact seeds, broken samples, and Allium macrostemonum / Allium chinense from different origins; at the same time, the product in the form of a kit can realize "one-stop" detection, which further expands the application scenarios and can cover the whole industry chain links such as traditional Chinese medicinal material planting bases, production enterprises, retail pharmacies, and regulatory agencies. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, a brief introduction will be given below to the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0022] Figure 1 Figure is an ITS2 sequence alignment chart of Allium macrostemonum and Allium chinense; Figure 2 Figure is an amplification chart of Allium macrostemonum and Allium chinense based on primer pair JP1; Figure 3 Figure is an amplification chart of Allium macrostemonum and Allium chinense based on primer pair JP2; Figure 4 Figure is an amplification chart of mixed samples of Allium macrostemonum and Allium chinense based on primer pair JP2. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0024] Example 1 1. Design of specific primers Based on the ITS2 sequences of Allium macrostemon and Allium fistulosum, comparative analysis found that there were different bases in three regions. Two groups of primers were designed near the markers. The primer sequence information is shown in Table 1.

[0025] Table 1 Specific primer pairs of Allium macrostemon and Allium fistulosum

[0026] 2. Identification of Allium macrostemon and Allium fistulosum 2.1 Instruments 2.1.1 Fluorescence PCR: StepOnePlus, purchased from Thermo Fisher Scientific, USA; TM 2.1.2 Refrigerated centrifuge: Sigma 3K15, purchased from Sigma, Germany; 2.1.3 Electronic balance: 1 / 100,000 electronic balance, model AB135-S, purchased from Mettler-Toledo.

[0027] 2.1.4 Dry thermostat: DC10, purchased from Hangzhou Ruicheng Instrument Co., Ltd.

[0028] 2.2 Reagents 2.2.1 Primers: synthesized by Shanghai Shengong Biological Co., Ltd.

[0029] 2.2.2 Nucleic acid extraction reagent: plant genomic DNA extraction kit (DP305) from Beijing Tiangeng Biochemical Co., Ltd. 2.2.3 PCR reagent: ChamQ Universal SYBR qPCR Master Mix, purchased from Nanjing Novozyme Biological Technology Co., Ltd., water: RNase-free double distilled water, purchased from Shanghai Shengong Biological Co., Ltd. 2.3 Nucleic acid extraction: 30 mg of Allium macrostemon and Allium fistulosum were weighed and ground into powder, and the sample genomic DNA was extracted according to the DP305 instructions.

[0030] 2.4 Fluorescence PCR amplification 2.4.1 PCR reaction system: DNA template 50 ng, SYBR qPCR Master Mix 10 ul, left and right primers each 1 ul, add sterile double distilled water to 20 uL.

[0031] 2.4.2 The fluorescence PCR instrument amplification program is shown in Table 2 Table 2 Fluorescence PCR instrument amplification program

[0032] ​2.4.3 PCR reaction: 2 groups of primers were used to amplify the DNA of Allium victorialis and Allium chinense samples.

[0033] 2.5 Sample identification analysis: The sample amplification results of primer pair JP1-F, JP1-R are shown in Figure 2 The sample amplification results of primer pair JP2-F, JP2-R are shown in Figure 3 The primer group JP1-F, JP1-R has an amplification curve, and is identified as Allium victorialis. The primer group JP2-F, JP2-R has an amplification curve, and is identified as Allium chinense.

[0034] Example 2 30 mg of Allium victorialis and Allium chinense mixed samples were weighed, and the sample amounts and mixing ratios are shown in Table 3. Table 3 Sample amounts and mixing ratios

[0035] DNA extraction: The same as in Example 1, except that the sample in this example is a mixture of Allium victorialis and Allium chinense.

[0036] Fluorescent PCR amplification: The same as in Example 1.

[0037] Sample identification analysis: The samples of primer pair JP1-F, JP1-R and primer pair JP2-F, JP2-R were amplified, and the results showed that the mixed samples of different proportions all had amplification curves, as shown in Figure 4 .

[0038] As can be seen from the examples, the present application can quickly identify Allium victorialis, Allium chinense, and whether Allium chinense is mixed in Allium victorialis by designing specific primers for ITS2 region. The entire detection process is short and does not require electrophoresis. The sample pretreatment does not involve toxic reagents, which is conducive to environmental protection and occupational health and safety. At the same time, the detection is greatly shortened by using a fluorescent PCR instrument. The method is simple, practical, fast and accurate, and is suitable for popularization and use.

[0039] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A specific primer for rapid identification of Chinese medicinal materials leek seeds and onion seeds, characterized in that: The invention comprises a specific primer pair JP1-F and JP1-R for identifying leek seeds and a specific primer pair JP2-F and JP2-R for identifying scallion seeds; the nucleotide sequence of the JP1-F is shown in the sequence list SEQ ID No: 1, the nucleotide sequence of the JP1-R is shown in the sequence list SEQ ID No: 2, the nucleotide sequence of the JP2-F is shown in the sequence list SEQ ID No: 3, and the nucleotide sequence of the JP2-R is shown in the sequence list SEQ ID No:

4.

2. A method for rapid identification of Chinese medicinal materials leek seeds and onion seeds, characterized in that: The steps include: (1) Extraction of total nucleic acid from samples: Extract the total nucleic acid from the Chinese medicinal materials to be tested; (2) Fluorescence PCR amplification: using the total nucleic acid of the sample to be tested as a template, fluorescence PCR amplification is performed using the specific primer pairs JP1-F / JP1-R and JP2-F / JP2-R described in claim 1; (3) Result determination: The sample type was determined by detecting the fluorescence signal. If only the specific primer pair JP1-F / JP1-R showed a fluorescence signal, the sample to be tested was leek seeds; if only the specific primer pair JP2-F / JP2-R showed a fluorescence signal, the sample to be tested was scallion seeds; if both the specific primer pairs JP1-F / JP1-R and JP2-F / JP2-R showed fluorescence signals, the sample to be tested was a mixture of leek seeds and scallion seeds.

3. The method for rapid identification of Chinese medicinal materials leek seeds and green onion seeds as claimed in claim 2, wherein: In step (1), a plant genomic DNA extraction kit is used to extract the total nucleic acid of the sample to be tested, and the sample to be tested needs to be ground into powder before extraction.

4. The method for rapid identification of the Chinese medicinal materials leek seeds and green onion seeds as claimed in claim 2, wherein: In step (2), the total volume of the reaction system for fluorescent PCR amplification is 20 μL, and the specific components include: 50 ng DNA template, 10 μL SYBR qPCR Master Mix, 1 μL JP1-F primer, 1 μL JP1-R primer, and the balance is sterile double-distilled water; or: 50 ng DNA template, 10 μL SYBR qPCR Master Mix, 1 μL JP2-F primer, 1 μL JP2-R primer, and the balance is sterile double-distilled water; the sterile double-distilled water is used to make up the reaction system to 20 μL.

5. The method for rapid identification of Chinese medicinal materials leek seeds and green onion seeds as claimed in claim 2, wherein: In step (2), the amplification program of fluorescent PCR is as follows: pre-denaturation at 95°C for 2 min; followed by 40 cycles, each cycle including denaturation at 95°C for 5 s and annealing and extension at 60°C for 30 s.

6. A kit for rapid identification of Chinese medicinal materials leek seeds and scallion seeds, characterized in that: The method comprises the specific primer pairs JP1-F / JP1-R and JP2-F / JP2-R according to claim 1, and reagents required for performing a fluorescent PCR reaction.

7. The kit for rapid identification of the Chinese medicinal materials leek seeds and scallion seeds according to claim 6, wherein: The reagents required for the fluorescent PCR reaction include ChamQ Universal SYBR qPCR Master Mix and RNase-free double-distilled water. The ChamQ Universal SYBR qPCR Master Mix was purchased from Nanjing Novozymes Biotechnology Co., Ltd., and the RNase-free double-distilled water was purchased from Shanghai Sangon Biotechnology Co., Ltd.

8. The kit for rapid identification of the Chinese medicinal materials leek seeds and scallion seeds according to claim 6, wherein: The kit also includes a sample grinding tool and a nucleic acid extraction auxiliary reagent, wherein the nucleic acid extraction auxiliary reagent is a lysis solution and a washing solution required in the process of extracting plant genomic DNA.

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