Method for identifying rhizoma acori graminei and herba lysimachiae as well as aqueous extract preparation thereof through multiple PCR (Polymerase Chain Reaction

Abstract

The invention relates to the technical field of traditional Chinese medicine identification, in particular to a primer pair, a kit, application of the primer pair in specific identification of rhizoma acori graminei and cattail traditional Chinese medicinal materials, traditional Chinese medicine formula granules and other aqueous extract preparations and an identification method. Primer pairs comprise a first primer pair and a second primer pair, the first primer pair comprises a forward primer SEQ ID NO: 1 and a reverse primer SEQ ID NO: 2, the second primer pair comprises a forward primer SEQ ID NO: 3 and a reverse primer SEQ ID NO: 4, the primer pairs are adopted for amplification, and the obtained product can be used for identifying species, authenticity and adulteration conditions of the Chinese medicinal materials of the typha angustifolia and the rhizoma acori graminei and water extract preparations of the typha angustifolia and the rhizoma acori graminei. The multiplex PCR identification method is good in specificity and high in result accuracy, can solve the problem that the water extract is difficult to identify after losing form, and provides guarantee for the medication safety and clinical curative effect of the rhizoma acori graminei and the herba lysimachiae.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine identification, and in particular to a method and application of multiplex PCR identification of Acorus tatarinowii and Taurus chinensis Chinese medicinal materials and their water extract preparations. Background Art

[0002] The Chinese Pharmacopoeia (2020 edition) stipulates that the source of Acorus gramineus is the dried rhizome of Acorus gramineus Schott, a plant of the Araceae family. It is dug in autumn and winter, the fibrous roots and mud are removed, and it is dried in the sun. It has a fragrant aroma, bitter and slightly spicy taste, and is mainly used to open the mind and eliminate phlegm, wake up the mind and improve intelligence, and eliminate dampness and stimulate the appetite. It is clinically used for amnesia, insomnia, tinnitus, deafness, coma, epilepsy, abdominal distension without hunger, and dysentery. Golden money is Acorus gramineus Soland, a plant of the Araceae family. Among them, Acorus xiangyeus, Acorus macrospadiceus, and Acorus latifolius are synonyms of Acorus gramineus. They are all officially named Golden Money Acorus gramineus in Plant Intelligence (Plant Intelligence Information System). The rhizome of Golden Money Cattail can be used as medicine, mainly for treating phlegm obstruction, unclear consciousness, chronic tracheitis, dysentery, enteritis, etc. "Shennong's Herbal Classic" records that Golden Money Cattail "treats wind, cold and dampness, cough, reverse breathing, opens the heart, nourishes the five internal organs, clears the nine orifices, brightens the ears and eyes, and produces sound."

[0003] Acorus calamus and golden money are closely related in evolution, but they are different species. Their chemical compositions and effects are different and they cannot be mixed. As the appearance of the two medicinal materials is very similar, the traditional identification method is limited by the professional knowledge and experience of the inspectors and is difficult to identify. In addition, after being processed into decoction pieces, the characteristics of the two herbs are lost, and it is difficult to accurately identify the mixed samples of Acorus calamus and golden money, which greatly affects the safety of Acorus calamus.

[0004] In order to ensure the safety and clinical efficacy of traditional Chinese medicine, it is necessary to develop an identification method for Acorus calamus and Taurus chinensis with high accuracy, good specificity and high detection efficiency. Summary of the invention

[0005] Based on this, the purpose of this application includes providing a specific identification method, specific identification primer pair, kit and application of Acorus calamus and Taurus chinensis Chinese medicinal materials and their water extract preparations. The specific identification primers are used to amplify the sample DNA, and the obtained amplification products can be used to identify the Acorus calamus and Taurus chinensis Chinese medicinal materials and their water extract preparations.

[0006] The purpose of this application can be achieved through the following technical solutions:

[0007] In one aspect of the present application, a primer pair is provided, the primer pair comprising a first primer pair and / or a second primer pair;

[0008] The first primer pair includes: a forward primer having a base sequence as shown in SEQ ID NO: 1 and a reverse primer having a base sequence as shown in SEQ ID NO: 2;

[0009] The second primer pair includes: a forward primer having a base sequence as shown in SEQ ID NO:3 and a reverse primer having a base sequence as shown in SEQ ID NO:4.

[0010] In another aspect of the present application, there is provided application of the primer pair described in the above technical solution in specific identification of Chinese medicinal materials Acorus tatarinowii and Taurus chinensis and their water extract preparations.

[0011] In another aspect of the present application, a kit is provided for specifically identifying the Chinese medicinal materials of Acorus tatarinowii and Taurus chinensis and their aqueous extracts, which comprises the primer pair described in any of the above technical solutions.

[0012] In some embodiments, the kit further comprises an amplification reagent.

[0013] In some embodiments, the amplification reagent includes one or more of an amplification buffer, dNTPs, and a DNA polymerase.

[0014] In another aspect of the present application, a multiplex PCR identification method for Chinese medicinal materials of Acorus tatarinowii and Taurus chinensis and their aqueous extracts is provided, comprising the following steps:

[0015] Extracting DNA from the sample to be tested;

[0016] Amplifying the DNA of the sample to be tested using the primer pair or kit described in any of the above technical solutions to prepare an amplification product; and,

[0017] The amplified product is detected, and the sample to be tested is identified based on the obtained detection result.

[0018] In some embodiments, the amplification procedure includes: pre-denaturing the amplified reaction system at 94°C to 96°C for 4min to 6min, then cycling 33 to 35 times under a preset program, and then extending at 71°C to 73°C for 4min to 6min;

[0019] The preset program includes: denaturation at 94° C. to 96° C. for 20 s to 40 s, annealing at 61° C. to 63° C. for 20 s to 40 s, and extension at 71° C. to 73° C. for 20 s to 40 s.

[0020] In some embodiments, each 25 μL of amplification reaction system includes: 2 μL to 3 μL of 10× PCR buffer, 1.5 μL to 2.5 μL of dNTP, 0.15 μL to 0.25 μL of Taq DNA polymerase, 0.2 μL to 0.4 μL of the first forward primer, 0.2 μL to 0.4 μL of the first reverse primer, 0.2 μL to 0.4 μL of the second forward primer, 0.2 μL to 0.4 μL of the second reverse primer and 2 μL to 4 μL of DNA of the sample to be tested.

[0021] In some embodiments, in the amplification system: the concentration of the first forward primer is 0.2 μmol / L to 0.4 μmol / L, the concentration of the first reverse primer is 0.2 μmol / L to 0.4 μmol / L, the concentration of the second forward primer is 0.2 μmol / L to 0.4 μmol / L, and the concentration of the second reverse primer is 0.2 μmol / L to 0.4 μmol / L.

[0022] In some embodiments, in the multiplex PCR identification method, the amplification products are detected, and the identification of the sample to be tested is achieved according to the obtained detection results, including:

[0023] The amplified product is detected by agarose gel electrophoresis, and whether the sample to be tested contains Acorus tatarinowii and / or Tamarindus chinensis is determined according to whether bands appear at 344 bp and / or 321 bp in the obtained electrophoresis diagram.

[0024] The present application provides a primer pair, which includes a first primer pair and / or a second primer pair; the first primer pair includes: a forward primer with a base sequence as shown in SEQ ID NO: 1 and a reverse primer with a base sequence as shown in SEQ ID NO: 2; the second primer pair includes: a forward primer with a base sequence as shown in SEQ ID NO: 3 and a reverse primer with a base sequence as shown in SEQ ID NO: 4. The primer pair is used to amplify the DNA of Chinese medicinal materials of Golden Money and Acorus Tatarinowii and their water extract preparations, and the obtained amplification products can be used to identify the species of Chinese medicinal materials of Golden Money and Acorus Tatarinowii and their water extract preparations, as well as their authenticity or adulteration, and can solve the problem that water extracts (including Chinese medicine formula granules) are difficult to identify after losing their morphology.

[0025] The identification method of the present application has good specificity and high result accuracy, which provides a guarantee for the medication safety and clinical efficacy of the Chinese medicinal materials of Golden Money and Acorus calamus and their water extract preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 The results of multiple primer screening of Acorus tatarinowii and Typha chinensis in one embodiment of the present application are as follows: Figure 1 In the figure, M refers to DNA maker DL 500, 1 and 2 are different samples of Acorus tatarinowii, 3 and 4 are different samples of Typha chinensis, and N is a blank sample (ddH2O);

[0028] Figure 2 This is the investigation result of amplification using different enzymes in one embodiment of the present application. Figure 2 In the figure, M refers to DNA makerDL 1000, 1, 2 and 3 are different samples of Acorus tatarinowii, 4, 5 and 6 are different samples of Typha chinensis, and N is a blank sample (ddH2O);

[0029] Figure 3 This is the investigation result of using different annealing temperatures after amplification in one embodiment of the present application. Figure 3 In the figure, M refers to DNAmaker DL 1000, 1, 2 and 3 are different samples of Acorus tatarinowii, 4, 5 and 6 are different samples of Typha chinensis, and N is a blank sample (ddH2O);

[0030] Figure 4 The results of the investigation of different cycle numbers used in the amplification procedure in one embodiment of the present application are as follows: Figure 4 In the figure, M refers to DNA maker DL 1000, 1, 2 and 3 are different samples of Acorus tatarinowii, 4, 5 and 6 are different samples of Typha chinensis, and N is a blank sample (ddH2O);

[0031] Figure 5 This is the result of the investigation of the detection limit of specific identification in an embodiment of the present application. Figure 5 In the table, M refers to DNA makerDL 1000, 1 refers to 1-fold dilution, 2 refers to 10-fold dilution, 3 refers to 100-fold dilution, 4 refers to 1000-fold dilution, 5 refers to 10000-fold dilution, and N refers to blank sample (ddH2O);

[0032] Figure 6 This is the result of the investigation on the applicability of specific identification in one embodiment of the present application. Figure 6In the table, M refers to DNA makerDL 1000, 1 to 4 are different samples of Acorus tatarinowii, 5 to 20 are different samples of Psoralea corylifolia, and N is a blank sample (ddH2O).

[0033] Figure 7 This is the test result of the suitability of the water extract of the specific identification method in one embodiment. Figure 7 In the figure, M refers to DNAmaker DL 1000, 1-7 are different mixed samples of Acorus tatarinowii and Tamarindus chinensis water extracts, and N is a blank sample (ddH2O). DETAILED DESCRIPTION

[0034] The present application will be further described below in conjunction with the embodiments, examples and accompanying drawings. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content taught in the present application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the scope of protection of the claims attached to the present application.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0036] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0037] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0038] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0039] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0040] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0041] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0042] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0043] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control.

[0044] In the present application, weight may be μg, mg, g, kg or other mass units known in the chemical industry.

[0045] The water extract products involved in this application include Chinese medicine formula granules and other water extract products other than Chinese medicine formula granules, and other water extract products other than Chinese medicine formula granules include Chinese medicine standard decoctions. The water extract products of this application mainly refer to Chinese medicine products prepared with water extracts of Chinese medicinal materials such as Golden Money or Acorus Tatarinowii, which are prepared by extracting Golden Money or Acorus Tatarinowii under heating conditions with water as the extraction water (for example, decocting with water).

[0046] The traditional PCR-based technical solution for identifying Acorus calamus is mainly limited to medicinal materials, with a small scope of application, and no identification method for Acorus calamus and Taurus chinensis Chinese medicinal materials or their water extracts has been reported. For example, CN105177131A discloses a specific primer pair and method for identifying Tibetan Acorus calamus and Acorus calamus, which analyzes the size of enzyme-cut fragments based on restriction fragment length polymorphism PCR technology (PCR-RFLP) to identify the authenticity of Tibetan Acorus calamus and Acorus calamus, but does not involve the identification of Taurus chinensis; CN115873931A discloses a method for detecting Acorus calamus, Acorus calamus, and Acorus tatarinowii based on multiplex ligation-dependent probe amplification technology. The method was used to identify the adulteration of Acorus calamus, Acorus tatarinowii and Acorus gramineus. The method did not involve the identification of Goldenrod. There are few reports on the adulteration of Acorus calamus and Goldenrod. In addition, there are few studies on the specific identification of the water extract of Acorus calamus.

[0047] To this end, in one aspect of the present application, a primer pair is provided, comprising a first primer pair and / or a second primer pair;

[0048] The first primer pair includes: a forward primer having a base sequence as shown in SEQ ID NO: 1 and a reverse primer having a base sequence as shown in SEQ ID NO: 2;

[0049] The second primer pair includes: a forward primer having a base sequence as shown in SEQ ID NO:3 and a reverse primer having a base sequence as shown in SEQ ID NO:4.

[0050] In some embodiments, the primer pair is used to amplify the DNA of Chinese medicinal materials of Golden Money and Acorus Tatarinowii and their water extracts to obtain amplified products, and it is found that the amplified product of Acorus Tatarinowii contains only a 344bp DNA fragment, the amplified product of Golden Money contains only a 321bp DNA fragment, and the mixed amplified product of the two contains both a 344bp DNA fragment and a 321bp DNA fragment. The detection result can be used for the specific identification of Chinese medicinal materials of Golden Money and Acorus Tatarinowii and their water extracts.

[0051] In another aspect of the present application, there is provided application of the primer pair described in the above technical solution in specific identification of Acorus tatarinowii and Taurus chinensis Chinese medicinal materials and their water extract preparations.

[0052] The specific primer pair of the present application can simultaneously identify Acorus tatarinowii and Taurus chinensis using the same amplification system and procedure, thereby achieving efficient and rapid identification from a molecular perspective.

[0053] Identification in this application includes: species identification, authenticity identification and adulteration identification.

[0054] The specific identification primer pair provided in the present application is used to amplify the DNA of the specific identification primer pair of the Chinese medicinal materials of Golden Money and Acorus Tatarinowii and their water extract preparations. The obtained amplification product can be used not only for the identification of Golden Money and Acorus Tatarinowii Chinese medicinal materials, but also for the identification of Chinese medicinal formula granules or other water extract products of Golden Money and Acorus Tatarinowii, solving the problem that the water extract products are difficult to identify after losing their morphology. The identification with the specific identification primer pair has good specificity and high accuracy, which provides a guarantee for the medication safety and clinical efficacy of Golden Money and Acorus Tatarinowii Chinese medicinal materials and their water extract preparations.

[0055] In another aspect of the present application, a kit is provided for specifically identifying the Chinese medicinal materials of Acorus tatarinowii and Taurus chinensis and their aqueous extracts, which comprises the primer pair described in any of the above technical solutions.

[0056] In some embodiments, the kit further comprises an amplification reagent.

[0057] In some embodiments, the amplification reagents include one or more of an amplification buffer, dNTPs, and a DNA polymerase.

[0058] In some embodiments, the amplification reagents include:

[0059] 1) Amplification buffer, including 10× PCR Buffer I;

[0060] 2) dNTP (deoxy-ribonucleoside triphosphate);

[0061] 3) DNA polymerase, including Taq enzyme;

[0062] 4) Water, further sterilized water.

[0063] In another aspect of the present application, a multiplex PCR identification method for Chinese medicinal materials of Acorus tatarinowii and Taurus chinensis and their aqueous extracts is provided, the identification method comprising the following steps:

[0064] Extracting DNA from the sample to be tested;

[0065] Amplifying the DNA of the sample to be tested using the primer pair or kit of the present application to prepare an amplification product; and,

[0066] The amplified product is detected, and the sample to be tested is identified based on the obtained detection result.

[0067] In some embodiments, during the amplification procedure, the annealing temperature is 61°C to 63°C.

[0068] In some embodiments, the number of cycles in the amplification procedure is 33 to 35 times.

[0069] In some embodiments, the amplification procedure includes: pre-denaturing the amplification system at 94°C to 96°C for 4min to 6min, then cycling 33 to 35 times under a preset program, and finally extending at 71°C to 73°C for 4min to 6min;

[0070] Furthermore, the preset program includes: denaturation at 94°C to 96°C for 20s to 40s, annealing at 61°C to 63°C for 20s to 40s, and extension at 71°C to 73°C for 20s to 40s.

[0071] In some embodiments, the amplification procedure includes: pre-denaturing the amplification reaction system at 95° C. for 5 min, cycling 34 times under a preset program, and then extending at 72° C. for 5 min;

[0072] Furthermore, the preset program includes: denaturation at 95°C for 30s, annealing at 62°C for 30s, and extension at 72°C for 30s.

[0073] In some embodiments, each 25 μL amplification reaction system includes: 2 μL to 3 μL of 10× PCR buffer, 1.5 μL to 2.5 μL of dNTPs, 0.15 μL to 0.25 μL of Taq DNA polymerase, 0.2 μL to 0.4 μL of a first forward primer, 0.2 μL to 0.4 μL of a first reverse primer, 0.2 μL to 0.4 μL of a second forward primer, 0.2 μL to 0.4 μL of a second reverse primer, 2 μL to 4 μL of a DNA template, and water.

[0074] In some embodiments, each 25 μL amplification reaction system includes: 2.5 μL of 10× PCR buffer, 2.0 μL of dNTP, 0.2 μL of Taq DNA polymerase, 0.3 μL of a first forward primer, 0.3 μL of a first reverse primer, 0.3 μL of a second forward primer, 0.3 μL of a second reverse primer, 3 μL of a DNA template, and 16.1 μL of water.

[0075] In some more specific embodiments, the total reaction volume of the multiplex PCR amplification system is 25 μL, including: 2.5 μL of 10×PCR buffer; 2.0 μL of dNTP (2.5 mmol / L); 0.2 μL of Ex Taq DNA polymerase (5 U / μL); 0.3 μL (10 μmol / L) of each of a forward primer with a base sequence as shown in SEQ ID NO: 1, a reverse primer with a base sequence as shown in SEQ ID NO: 2, a forward primer with a base sequence as shown in SEQ ID NO: 3, and a reverse primer with a base sequence as shown in SEQ ID NO: 4; 3.0 μL of DNA template; and the volume is filled to 25 μL with high-pressure sterilized ultrapure water.

[0076] In some more specific embodiments, the steps of multiple PCR amplification include: oscillating and mixing the reaction solution, and centrifuging it momentarily; placing the reaction mixture system on a PCR instrument for amplification, and the amplification program is: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 34 cycles; and finally extension at 72°C for 5 minutes.

[0077] Furthermore, after the amplification, 5 μL of 6× loading buffer was added to the reaction system, mixed, and the mixed product (6 μL to 8 μL) was spotted on a 2.5% agarose gel, electrophoresed at a voltage of 150 V for 35 min, developed with GelRed, and finally observed and recorded the results on a gel imager.

[0078] In some embodiments, the concentration of the first forward primer is 0.2 μmol / L to 0.4 μmol / L, the concentration of the first reverse primer is 0.2 μmol / L to 0.4 μmol / L, the concentration of the second forward primer is 0.2 μmol / L to 0.4 μmol / L, and the concentration of the second reverse primer is 0.2 μmol / L to 0.4 μmol / L.

[0079] In some embodiments, in the identification method, the amplification product is detected, and the identification of the sample to be tested is achieved according to the obtained detection result, including:

[0080] The amplified product was detected by agarose gel electrophoresis, and whether the sample to be tested contained Acorus tatarinowii and / or Taurus chinensis was determined according to whether a band appeared at 344 bp and / or 321 bp in the obtained electrophoresis diagram.

[0081] The following are some specific embodiments.

[0082] The following examples are given for the purpose of illustrating various embodiments of the present application, and are not intended to limit the present application in any way. Those skilled in the art will understand that the variations and other uses thereof are included within the spirit of the present application as defined by the scope of the claims. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources, and the promoter and terminator sequences mentioned in the examples can also be downloaded from NCBI, and the specific sequence start position can be obtained based on the primers in the primer table.

[0083] Example 1

[0084] 1. Primer list (Table 1)

[0085] Table 1

[0086]

[0087] 2. Sample list (Table 2)

[0088] Table 2

[0089]

[0090] 3. Extraction of genomic DNA

[0091] The genomic DNA of Acorus tatarinowii and Glechoma longituba was extracted using a high-efficiency plant genomic DNA extraction kit (catalog number: DP350-0, manufacturer: Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0092] Take 20 mg of medicinal powder, add 400 μL of buffer FGA and 6 μL of RNase A (10 mg / mL), vortex for 1 min, and leave at room temperature for 10 min. Add 130 μL of buffer LP2, mix thoroughly, and vortex for 1 min. Centrifuge at 12000rpm (~13400×g) for 5 min, and transfer the supernatant to a new centrifuge tube. Add 1.5 times the volume of buffer LP3, and immediately vortex and mix thoroughly for 15 seconds. Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000rpm for 30 seconds, pour out the waste liquid, and place the adsorption column CB3 in a collection tube.

[0093] Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, place the adsorption column CB3 in the collection tube, and repeat this step twice. Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 minutes, and discard the waste liquid. Leave the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual rinse solution in the adsorption material. Transfer the adsorption column CB3 to a clean centrifuge tube, add 50 μL of elution buffer TB to the middle part of the adsorption membrane, leave it at room temperature for 2 to 5 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution in a centrifuge tube.

[0094] The water extract of Acorus tatarinowii was extracted by the two-step CTAB method. Take 100 mg of freeze-dried powder of the water extract, add 1.2 mL of CTAB precipitation solution, vortex mix, place in a 65°C metal bath and incubate for 60 minutes, flip and mix 2 to 3 times during the period. Centrifuge at 12000 rpm for 5 minutes, discard the supernatant, add 1.0 mL of CTAB precipitation solution, vortex and mix, incubate at 65°C for 30 minutes, flip 3 to 5 times during the period. Centrifuge at 12000 rpm for 5 minutes, discard the supernatant, add 1.2 mL of CTAB extraction solution, 20 μL of proteinase K and 20 μL of β-mercaptoethanol, vortex and mix, incubate at 56°C overnight, flip and mix 2 to 3 times during the period. Add an equal volume of chloroform-isoamyl alcohol mixture (chloroform:isoamyl alcohol = 24:1), mix thoroughly to a milky white color, centrifuge at 4°C, 12000 rpm for 10 minutes, and repeat the operation twice. Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol and 10μL of a trace DNA precipitant, a white flocculent precipitate will appear, shake well, and refrigerate at -20℃ for 60min or overnight. Centrifuge at 12000rpm for 5min, discard the supernatant, the precipitate is DNA, add 700μL of 75% ethanol, flip several times to wash the DNA, repeat the operation 2 times. Add 700μL of anhydrous ethanol for washing, centrifuge at 12000rpm for 5min, discard the supernatant, place in a 37℃ metal bath to dry for 5min~10min, until the white clumps can fall off the tube wall, add 15μL of buffer TB.

[0095] 4. DNA Concentration Determination

[0096] Take the above DNA samples, use BioSpec-nano micro-volume UV spectrophotometer to determine the DNA concentration, record OD260 / OD230, OD260 / OD280 at the same time, and adjust the concentration to the corresponding concentration.

[0097] 5. Multiplex PCR Identification

[0098] (1) Primer design

[0099] According to the ITS2 sequences of Acorus calamus and Golden Taurus obtained by sequencing, the sequences were homologously aligned using BioEdit software. After proofreading, the specific SNP sites of Acorus calamus and Golden Taurus were analyzed. The base sequences containing the SNP sites were imported into Primer Premier 5 software for primer design. The detailed information is shown in Table 3. The primers were synthesized by Shanghai Biotech Co., Ltd.

[0100] Table 3 Primer information

[0101]

[0102] (2) Primer screening

[0103] The multiple PCR amplification system and procedure of Acorus tatarinowii and Taurus chinensis designed in this patent were established, as shown in Table 4:

[0104] Table 4 Multiple PCR reaction system of Acorus tatarinowii and Typha chinensis (25 μL)

[0105]

[0106]

[0107] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 30 s, for a total of 34 cycles; and finally extension at 72°C for 5 min.

[0108] PCR amplification was performed with the primers shown in Table 4. After the PCR amplification reaction was completed, 5 μL of 6× loading buffer was added to the reaction system and mixed. 6 μL to 8 μL of the mixed product was spotted on a 2.5% agarose gel and electrophoresed at 150 V for 35 min. The gel was developed with GelRed and the results were observed on a gel imager. Figure 1 . The results showed that no reaction bands appeared in the blank controls, indicating that the PCR reaction process was pollution-free and the results were reliable. Among them, the multiple primer pairs SCP-8 and JQP-6 designed in this patent can specifically amplify Acorus calamus and Taurus chinensis. The target band lengths of Acorus calamus obtained by PCR amplification were 344bp and 321bp for Taurus chinensis. However, some samples had non-specific amplification, which could be optimized through the later PCR reaction conditions. This shows that the primer set can specifically detect Acorus calamus and Taurus chinensis, and can be used as multiple specific primers for rapid identification of Acorus calamus and Taurus chinensis.

[0109] (3) Optimization of PCR reaction conditions

[0110] 1) Investigation of different enzymes

[0111] The effects of DNA polymerases 2×SanTaq, Ex Taq, SpeedSTAR, and PremixTaq on PCR amplification were investigated by single factor. Figure 2 As shown, when Ex Taq and 2×SanTaq polymerases were used, single bands of Acorus tatarinowii and Tachys chinensis could be obtained, and both polymerases can be used. Among them, this patent selected Ex Taq polymerase as the DNA polymerase for this time.

[0112] 2) Annealing temperature investigation

[0113] The effects of annealing temperatures of 58°C, 60°C, 62°C, and 64°C on PCR amplification were investigated by single factor analysis. Figure 3 As shown, when the annealing temperature is 58℃, the PCR band is the brightest, but non-specific amplification is obvious; at 60℃~62℃, the band brightness is moderate and the band is single; but in the early investigation, it was found that at 60℃, the Acorus tatarinowii primers would have non-specific amplification for some Goldenrod samples. In order to ensure the accuracy of the results, the annealing temperature was selected as 62℃.

[0114] 3) Investigation of different cycle numbers

[0115] The effects of 30, 32, 34, and 36 cycles on PCR amplification were investigated by single factor. Figure 4 As shown, with the increase of cycle number, the brightness of PCR amplification band gradually increased, and no false positive appeared in the blank control; when the cycle number was 34, the brightness of PCR band was moderate. In order to ensure the accuracy of the results, the cycle number was selected as 34.

[0116] (4) Detection limit and applicability of multiple PCR method for Acorus calamus

[0117] The sensitivity of the multiplex PCR method for Acorus tatarinowii was investigated. One batch of Acorus tatarinowii and one batch of Taurus chinensis were selected and diluted 1, 10, 100, 1000, and 10000 times, respectively. The concentration of the DNA template was determined and amplified according to the multiplex PCR system and amplification conditions of Acorus tatarinowii. Figure 5 As shown, specific bands can appear when the DNA of Acorus calamus slices is 9.3ng / μL and 1.86ng / μL, and when the DNA of Glehnia littoralis slices is 10.3ng / μL and 2.03ng / μL, and no false positives appear in the blank controls to ensure the brightness of the bands. It can be seen that the detection limit of Acorus calamus multiplex PCR identification is 1.86ng / μL.

[0118] Table 5 DNA dilution concentration

[0119]

[0120] To investigate the applicability of the multiplex PCR method for Acorus tatarinowii, 4 batches of Acorus tatarinowii medicinal materials, 16 batches of Taurus chinensis medicinal materials DNA, and 7 batches of mixed water extract DNA (the mixing ratio of Acorus tatarinowii and Taurus chinensis water extract DNA was 1:1) were selected, and amplification was performed according to the multiplex PCR system and amplification conditions of Acorus tatarinowii to verify the applicability of the multiplex PCR method for Acorus tatarinowii. Figure 6 As shown in the figure, the identification rate of Acorus calamus and Taurus tatarinowii by this method was 100%, the true positive rate was 100%, the true negative rate was 100%, the false positive rate was 0%, and the false negative rate was 0%. Figure 7 As shown, the mixed water extract DNA samples showed bands at 344 bp and 321 bp, respectively, indicating that this method can be used for multiplex PCR identification of Acorus tatarinowii and Taurus chinensis raw materials and water extracts.

[0121] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0122] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0123] The above-described embodiments only express several implementation methods of the present application, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can also be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided in the present application through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the attached claims, and the description and drawings may be used to explain the contents of the claims.

Claims

1. A primer pair, characterized in that: The primer pair includes a first primer pair and / or a second primer pair; The first primer pair includes: a forward primer having a base sequence as shown in SEQ ID NO: 1 and a reverse primer having a base sequence as shown in SEQ ID NO: 2; The second primer pair includes: a forward primer having a base sequence as shown in SEQ ID NO:3 and a reverse primer having a base sequence as shown in SEQ ID NO:

4.

2. Use of the primer pair described in claim 1 in the specific identification of Chinese medicinal materials such as Acorus tatarinowii and Taurus chinensis and their water extracts.

3. A kit for specific identification of Chinese medicinal materials of Acorus calamus and Tamarindus chinensis and their aqueous extracts, characterized in that: It comprises the primer pair according to claim 1.

4. The kit according to claim 3, characterized in that Amplification reagents are also included.

5. The kit according to claim 4, characterized in that The amplification reagent includes one or more of an amplification buffer, dNTP and a DNA polymerase.

6. A multiplex PCR identification method for Chinese medicinal materials of Acorus calamus and Tamarindus chinensis and their aqueous extracts, characterized in that: The following steps are involved: Extracting DNA from the sample to be tested; Amplifying the DNA of the sample to be tested using the primer pair described in claim 1 or the kit described in claim 2 or 3 to prepare an amplification product; as well as, The amplified product is detected, and the sample to be tested is identified based on the obtained detection result.

7. The multiplex PCR identification method according to claim 6, characterized in that: The amplification procedure includes: pre-denaturing the amplified reaction system at 94°C to 96°C for 4min to 6min, then cycling 33 to 35 times under a preset program, and then extending at 71°C to 73°C for 4min to 6min; The preset program includes: denaturation at 94° C. to 96° C. for 20 s to 40 s, annealing at 61° C. to 63° C. for 20 s to 40 s, and extension at 71° C. to 73° C. for 20 s to 40 s.

8. The multiplex PCR identification method according to claim 6, characterized in that: Each 25 μL amplification reaction system includes: 2 μL to 3 μL of 10×PCR buffer, 1.5 μL to 2.5 μL of dNTP, 0.15 μL to 0.25 μL of Taq DNA polymerase, 0.2 μL to 0.4 μL of the first forward primer, 0.2 μL to 0.4 μL of the first reverse primer, 0.2 μL to 0.4 μL of the second forward primer, 0.2 μL to 0.4 μL of the second reverse primer and 2 μL to 4 μL of DNA of the sample to be tested.

9. The multiplex PCR identification method according to claim 8, characterized in that: In the amplification system: the concentration of the first forward primer is 0.2 μmol / L to 0.4 μmol / L, the concentration of the first reverse primer is 0.2 μmol / L to 0.4 μmol / L, the concentration of the second forward primer is 0.2 μmol / L to 0.4 μmol / L, and the concentration of the second reverse primer is 0.2 μmol / L to 0.4 μmol / L.

10. The multiplex PCR identification method according to any one of claims 6 to 9, characterized in that: Detecting the amplified product and identifying the sample to be tested according to the obtained detection result, including: The amplified product is detected by agarose gel electrophoresis, and whether the sample to be tested contains Acorus tatarinowii and / or Tamarindus chinensis is determined according to whether bands appear at 344 bp and / or 321 bp in the obtained electrophoresis diagram.

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