WO2023134738A1

WO2023134738A1 - Specific identification primer pair for trionycis carapax and decoction pieces, traditional chinese medicine dispensing granules and other water extract products thereof, use of specific identification primer pair, and identification method

Info

Publication number: WO2023134738A1
Application number: PCT/CN2023/072028
Authority: WO
Inventors: 罗宇琴; 李国卫; 谭斯尹; 刑菊玲; 宋叶; 魏梅; 陈向东; 孙冬梅
Original assignee: 广东一方制药有限公司
Priority date: 2022-01-14
Filing date: 2023-01-13
Publication date: 2023-07-20
Also published as: CN114350819A

Abstract

The present application relates to a specific identification primer pair for Trionycis carapax and decoction pieces, traditional Chinese medicine dispensing granules and other water extract products thereof, the use of the specific identification primer pair, and an identification method. The specific identification primer pair comprises a forward primer as shown in SEQ ID NO: 1, and a reverse primer as shown in SEQ ID NO: 2.

Description

The specific identification primer pair of turtle shell and its decoction pieces, traditional Chinese medicine formula granules and other water extract products and its application and identification method

related application

This application requires an application filed on January 14, 2022, the application number is 202210044243X, and the name is "Tiejia and its decoction pieces, Chinese medicine formula granules and other water extract products. Specific identification primer pairs and their applications and identification methods" China Priority of Patent Application, which is hereby incorporated by reference in its entirety.

technical field

The invention relates to the technical field of quality detection of traditional Chinese medicines, in particular to a pair of specific identification primers for turtle shells and their decoction pieces, traditional Chinese medicine formula granules and other water extract products, and their application and identification method.

Background technique

According to the 2020 edition of the Chinese Pharmacopoeia, the turtle shell is derived from the carapace of the soft-shelled turtle (Trionyx sinensis Wiegmann). Turtle shell, first recorded in the "Shen Nong's Materia Medica" in the Han Dynasty, is listed as a middle grade. It has been recorded in successive dynasties of herbal medicines. It has the effects of nourishing yin and subsidizing yang, reducing fever and steaming, softening and resolving hard masses, and is often used for fever due to yin deficiency and steamy bones. Heat, deficiency of yin and hyperactivity of yang, dizziness, internal movement of deficiency wind, and dysentery of hands and feet. Modern pharmacological studies have confirmed that turtle shell has anti-tumor, immune regulation, anti-cirrhosis, and liver protection effects.

Turtle shell is a commonly used traditional Chinese medicine. It is more common to use the same genus and its counterfeit products as turtle shell in the market, such as Chinemys reevesii (Gray), Apalone spinifera (Apalone spinifera) and Florida soft-shelled turtle (Apalone ferox) , Trionyx steindachneri, Lissemys punctata, Trionyx hurum, Flat soft-shelled turtle (also known as Malayan soft-shelled turtle, Dogania subplana), Pelochelys bibroni, Giant soft-shelled turtle ( Trionyx gangeticus) A, African soft-shelled turtle (Trionyx triunguis) A, etc., they are close in character and shape, relying on traditional identification methods is difficult, and the identification accuracy is low, especially processed by vinegar and made into traditional Chinese medicine by water extraction After the standard decoction is further processed into traditional Chinese medicine formula granules, it is more difficult to carry out effective identification due to the loss of medicinal properties. In order to ensure the quality and curative effect of Biejiao in clinical use, it is very necessary to accurately and quickly identify the water extract products of Biejiao and its decoction pieces (including standard Chinese medicine decoction and Chinese medicine formula granules).

Contents of the invention

Based on this, according to various embodiments of the present application, a specific identification primer pair for turtle shells and its decoction pieces, Chinese medicine formula granules and other water extract products is provided, and the technical scheme is as follows:

A pair of specific identification primers for turtle shells and its decoction pieces, traditional Chinese medicine formula granules and other water extract products, the specific identification primer pair includes a forward primer and a base sequence as shown in SEQ ID NO:1 The reverse primer whose sequence is shown in SEQ ID NO:2.

A specific identification kit for turtle shell and its decoction pieces, traditional Chinese medicine formula granules and other water extract products, the reagent The box contains said pair of specific identification primers, and amplification reagents.

In one of the embodiments, the amplification reagent comprises amplification buffer, dNTP, DNA polymerase and water; the amplification buffer is 10×PCR Buffer I, and the DNA polymerase is Taq enzyme.

A specific identification method for turtle shells, turtle shell decoction pieces, turtle shell traditional Chinese medicine formula granules or other turtle shell water extract products, said specific identification method comprising the following steps:

Extract the DNA of the sample to be tested;

Using the pair of specific identification primers or the specific identification kit to amplify the DNA, separate the amplified products obtained, and carry out specific identification on the sample to be tested according to the obtained separated products.

In one of the embodiments, the specific identification of the sample to be tested according to the obtained separation product includes:

If the isolated product comprises a 236bp fragment, it means that the sample to be tested comprises turtle shells,

If the isolated product does not contain a 236bp fragment, it means that the sample to be tested contains fake turtle shells or does not contain turtle shells.

In one of the embodiments, the counterfeit turtle shell includes one or more of the turtle shell, horned turtle shell, Florida turtle shell and Shan Rui shell shell.

In one of the embodiments, the other turtle shell aqueous extract products include the standard decoction of turtle shell traditional Chinese medicine.

In one embodiment, the annealing temperature of the amplification is 59°C-61°C.

In one embodiment, the amplification reaction system includes: 2.5 μL of 10×PCR Buffer I containing Mg ²⁺ , 2.5 mmol/L dNTP, 10 μmol/L forward primer, 10 μmol/L reverse primer, 5 U/μL Taq enzyme, 30ng~50ng template DNA and 19.2μL water.

In one embodiment, the amplification reaction program includes: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, a total of 33 cycles; and finally 72°C extension for 5 min.

Details of one or more embodiments of the application are set forth in the following description, and other features, objects, and advantages of the application will become apparent from the description and claims hereof.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application or the conventional technology, the following will briefly introduce the accompanying drawings that need to be used in the description of the embodiments or the traditional technology. Obviously, the accompanying drawings in the following description are only the present invention For the embodiments of the application, those skilled in the art can also obtain other drawings based on the disclosed drawings without creative effort.

Fig. 1 is the result of an embodiment of the present invention according to the primers in the references to test the turtle shell and counterfeit samples; in Fig. 1, M.DNA maker DL 1000, 1. Turtle shell medicinal material G2003054, 2. Turtle shell medicinal material G2003056 , 3. Tortoise shell herbal medicine G1810179, 4. Tortoise shell herbal medicine G1810182, 5. Florida soft-shelled turtle medicine G2011026, 6. Florida soft-shelled turtle medicine G2011027, 7. Shanrui soft-shelled turtle medicine G2011028, 8. Blank (ddH ₂ O);

Fig. 2 is the result of detecting different detection objects by using specific identification primer pairs in an embodiment of the present invention; in Fig. 2, M.DNA maker DL 1000, 1. Turtle shell control medicinal material, 2-16. Turtle shell medicinal material, 17 -18. Vinegar turtle shell decoction pieces, 19-25. Turtle shell standard decoction, 26-27. Vinegar turtle shell standard decoction, 28-30. Turtle shell formula granules, 31-32. Vinegar turtle shell formula granules, 33- 34. Tortoise shell medicinal material, 35-36. Horned soft-shelled turtle medicinal material, 37-38. Florida soft-shelled turtle medicinal material, 39. Shanrui soft-shelled soft-shelled turtle medicinal material, N. Blank (ddH ₂ O).

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the application with reference to the drawings in the embodiments of the application. Apparently, the described embodiments are only some of the embodiments of the application, not all of them. Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the scope of protection of this application.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs. The terms used herein in the specification of the application are only for the purpose of describing specific embodiments, and are not intended to limit the application.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not intended to limit the invention in any way. Those skilled in the art will appreciate that changes and other uses therein are included within the spirit of the invention, as defined by the scope of the claims. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. The promoter and terminator sequences mentioned in the examples can also be downloaded from NCBI. The starting position of the specific sequence can be found in Learned from the primers in the primer table. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.

In the present invention, "the first aspect", "the second aspect" and so on are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance or quantity, nor as implying the importance or importance of the indicated technical features. quantity.

In the present invention, the technical features described in open form include closed technical solutions consisting of the enumerated features, as well as open technical solutions including the enumerated features.

The percentage content involved in the present invention, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid phase mixing, and refers to volume percentage for liquid-liquid phase mixing.

The percentage concentration involved in the present invention refers to the final concentration unless otherwise specified. The final concentration refers to the proportion of the added component in the system after the component is added.

The temperature parameters in the present invention, unless otherwise specifically limited, allow either constant temperature treatment or treatment within a certain temperature range. The isothermal treatment allows the temperature to fluctuate within the precision of the instrument control.

The water extract product involved in the present invention mainly refers to the traditional Chinese medicine product prepared from the water extract of the turtle shell or the decoction pieces of the turtle shell. ) to the turtle shell or turtle shell decoction pieces Line extraction preparations are obtained. The water extract products in the present invention include standard decoctions and traditional Chinese medicine formula granules.

The traditional PCR-based technical solutions for the identification of soft-shelled turtles are mainly limited to the identification of soft-shelled turtle shells medicinal materials and soft-shelled turtle shells. For example: CN108018362A discloses a set of specific primers for identifying soft-shelled turtles. This set of specific primers is designed for soft-shelled turtles and soft-shelled turtle medicinal materials. Compared with publicly known specific primers, it can distinguish different species of the same suborder. The identification of counterfeit products can be achieved; in addition, the amplified target product is ≤120bp, but the conditions are applicable to the original turtle methyl, medicinal materials, decoction pieces and their adulterated products. Another example: CN103074433A discloses a turtle shell DNA detection kit and an identification method based on the kit. The kit is mainly aimed at medicinal materials, and the genuine turtle shell is identified based on the simultaneous appearance of 1000bp and 500bp bands. However, there is still a technical gap in the use of PCR to identify water extracts of turtle shells and their decoction pieces.

Compared with using PCR technology to identify the medicinal materials and decoction pieces of turtle shells, it is much more difficult to use PCR technology to identify the water extract products and formula granules of turtle shells and their decoction pieces. The specific PCR identification of traditional Chinese medicine standard decoction and traditional Chinese medicine formula granules has not been reported yet. In this case, it is often difficult to obtain ideal results by referring to traditional DNA sample design primers and amplification methods. Therefore, it is difficult to apply the PCR identification method for the medicinal materials and decoction pieces of the turtle shell/vinegar turtle shell to the corresponding traditional Chinese medicine standard decoction and traditional Chinese medicine. Formula granules.

For this reason, the contriver has provided a kind of specific identification primer pair, uses this primer to amplify the DNA of the to-be-tested turtle shell/Veteria turtle water extract product such as Chinese medicine standard decoction and Chinese medicine formula granules, and the resulting amplified product contains a DNA fragment of about 236bp, while the water extract product corresponding to the counterfeit turtle shell does not contain the DNA fragment of about 236bp. Based on this result, it can be identified whether the water extract product of the turtle shell/vinegar turtle shell to be tested is Turtle shell/Vinegar turtle shell water extract products are authentic. At the same time, the specific identification primers of the present application are also suitable for raw material identification of turtle shells/vinegar turtle shells.

In the first aspect, the present invention provides a pair of specific identification primers for turtle shells and its decoction pieces and water extract products, the pair of specific identification primers includes a forward primer with a base sequence as shown in SEQ ID NO: 1 and The base sequence is the reverse primer shown in SEQ ID NO:2.

The annealing temperature of the primer pair provided by the invention is 59°C-61°C.

The specific identification primers provided by the present invention are used to amplify the DNA of the standard Chinese medicine decoction and other water extracts of the turtle shell, and the obtained amplification products can not only be used for the identification of the turtle shell and its decoction pieces, but also for the soft-shelled turtle. The identification of water extract products of A and its decoction pieces (including standard Chinese medicine decoction and Chinese medicine formula granules) distinguishes them from counterfeit water extract products, and solves the problem that water extract products are difficult to identify after losing their form. problem. The specific identification primer pair is used for identification, which has good specificity and high accuracy of results, and provides guarantee for the drug safety and clinical efficacy of the standard Chinese medicine decoction or other water extract products of the turtle shell and its decoction pieces.

In the second aspect, the present invention provides a specific identification kit for turtle shells and its decoction pieces, traditional Chinese medicine formula granules and other water extract products, said kit comprising the specific identification primer pair described in claim 1, and an extension Reagents.

In one example, the amplification reagent comprises an amplification buffer, dNTPs, DNA polymerase and water; the The amplification buffer is 10×PCR Buffer I, and the DNA polymerase is Taq enzyme. It can be understood that the water in the amplification reagent of the present invention is sterilized water.

In the third aspect, the present invention provides a specific identification method for turtle shells, turtle shell decoction pieces, turtle shell traditional Chinese medicine formula granules, or other turtle shell water extract products. The specific identification method includes the following steps:

Extract the DNA of the sample to be tested;

In the specific identification method provided by the present invention, the specific identification of the sample to be tested according to the obtained separation product includes:

The counterfeit turtle shells described in the present invention can be, but not limited to: tortoise shells, horned turtle shells, Florida turtle shells, and Shanrui shell shells.

In one example, the other turtle shell aqueous extract products include the standard decoction of turtle shell traditional Chinese medicine.

In one example, the annealing temperature of the amplification is 59°C-61°C.

In one example, the amplification reaction system contains: 2.5 μL 10×PCR Buffer I containing Mg ²⁺ , 2.5 mmol/L dNTP, 10 μmol/L forward primer, 10 μmol/L reverse primer, 5 U/μL Taq Enzyme, 30ng～50ng template DNA and 19.2 μL water.

In one example, the amplification reaction program includes: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, a total of 33 cycles; and finally 72°C extension for 5 min.

In one example, the sample to be tested is a turtle shell-like sample or a turtle shell decoction piece-like sample, and the DNA is extracted using the SDS-proteinase K method.

In one example, the sample to be tested is a water extract product, the DNA is extracted using the CTAB method, and proteinase K is added to the CTAB extract used in the CTAB method. As mentioned above, the water-extracted product of the present invention refers to a product made from turtle shells or turtle shell decoction pieces through decoction and other heating processes, which can be selected from, including but not limited to: turtle shell traditional Chinese medicine standard decoction , Biejia Traditional Chinese Medicine Formula Granules, Vinegar-Biejia Chinese Medicine Standard Decoction and Vinegar-Biejia Traditional Chinese Medicine Formula Granules. During the preparation process of these water-extracted products, the structure is destroyed due to heating, and the degree of DNA degradation is similar to that of ancient DNA samples. By adopting the method for extracting DNA of the present invention, the required amount of DNA can be extracted from the damaged DNA to be tested for subsequent amplification. Moreover, during the DNA extraction process using the CTAB method, the interference of the excipients contained in the traditional Chinese medicine formula granules on the DNA extraction can be effectively avoided.

The decoction pieces described in the present invention can be, but not limited to, the turtle shell.

The water-extracted product of the present invention refers to a product made from turtle shells or turtle shell decoction pieces through decoction and other heating processes, which can be selected from, including but not limited to: turtle shell traditional Chinese medicine standard decoction, turtle shell traditional Chinese medicine formula Granules, Vinegar-Biejia Traditional Chinese Medicine Standard Decoction and Vinegar-Biejia Traditional Chinese Medicine Formula Granules.

Example 1

1. Sample list

Table 1

2. Extraction of DNA from medicinal materials, freeze-dried powder and traditional Chinese medicine formula granules

(1) DNA extraction of medicinal materials and decoction pieces

Take 0.05g of turtle shell medicinal materials, vinegar slices of turtle shell and common counterfeit products, grind them into powder, put them in a 2mL centrifuge tube, add 1.0mL of SDS lysate (50mmol/L Tris-HCl, 200mmol/L NaCl, 250mmol/L EDTA , 1% SDS) and 10 μL of proteinase K, vortexed to mix well, and incubated at 56° C. for 4 hours to 6 hours.

Take it out, cool it to room temperature, add an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1), mix thoroughly until it becomes milky white, centrifuge at 4°C (12,000 rpm) for 10 minutes, and absorb 800 μL to 1000 μL of the supernatant In a new 2mL centrifuge tube, repeat the operation once from "adding an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1)". Aspirate 600 μL-800 μL of the supernatant, and repeat the operation once from “adding an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1)”. Pipette 400 μL to 600 μL of the supernatant into a 1.5 mL centrifuge tube, add an equal volume of isopropanol, and let stand at minus 20°C for 60 to 90 minutes. Take it out, centrifuge (rotating at 12,000 rpm) for 5 minutes, discard the supernatant, wash the precipitate with 75% (v/v) ethanol three times, then wash the precipitate once with absolute ethanol, and incubate at 37°C for 30 minutes , After the ethanol volatilizes, add 50 μL of sterilized water to dissolve, as the test solution, store at 4°C or store at minus 20°C for a long time.

(2) DNA extraction of standard Chinese medicine decoction and Chinese medicine formula granules

Take 0.1 g of the freeze-dried powder and formula granules of vinegar turtle, grind them into powder, put them in a 2 mL centrifuge tube, add 1.2 mL of CTAB precipitation solution preheated at 56 °C, mix well, heat in a water bath at 56 °C for 60 min, and cool to room temperature. Centrifuge at 12000r/min for 5min, discard the supernatant, then add 1.2mL of CTAB precipitation solution and operate in the same way. Add 900 μL CTAB extract, 10 μL proteinase K, and 10 μL β-mercaptoethanol to the centrifuge tube in sequence, mix well, and heat in a water bath at 65°C for 120 min. Take out the centrifuge tube, add an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1) after cooling to room temperature, vortex and mix well, centrifuge at 12000r/min, 4°C for 10min, take 800μL of the supernatant and add a new 2mL In the centrifuge tube, repeat the operation once, and then take 600 μL of the supernatant into a 1.5 mL centrifuge tube, add an equal volume of isopropanol or isopropanol-3mol/L sodium acetate, and let stand at -20°C for 60 min. Take out the centrifuge tube and centrifuge at 12000r/min for 5min, discard the supernatant, wash the pellet three times with 75% ethanol and two times with absolute ethanol, centrifuge at 12000r/min for 5min, discard the supernatant, and incubate the pellet at 37°C After 30 minutes, after the ethanol has evaporated, add 30 μL of sterilized water to dissolve, store at 4°C or store at minus 20°C for a long time.

(3) DNA concentration measurement

Take the above DNA samples, measure DNA concentration with BioSpec-nano micro-volume UV spectrophotometer, record OD260/OD230, OD260/OD280 at the same time, and adjust the concentration to 50ng/μL～100ng/μL.

3. Identification

(1) Sequence analysis and primer design

According to the primers in the references, the samples of turtle shell and counterfeit products were tested, and the results are shown in Figure 1. In Figure 1, M.DNA maker DL 1000, 1. Turtle shell medicinal material G2003054, 2. Turtle shell medicinal material G2003056, 3. Tortoise shell medicinal material G1810179, 4. Tortoise shell medicinal material G1810182, 5. Florida soft-shelled turtle medicinal material G2011026, 6. Florida soft-shelled soft-shelled soft-shelled medicinal material G2011027, 7. Shanrui soft-shelled soft-shelled medicinal material G2011028, 8. Blank (ddH ₂ O).

Note: The literature primers are from [1] Cheng Suqian, Yuan Yuan, Liu Fuyan, et al. Specific PCR method to identify the medicinal materials and decoction pieces of turtle shell [J]. Chinese Journal of Traditional Chinese Medicine, 2018, 43(23): 4569-4574.

[2] Li Nan, Yu Pingtian, Jiao Zhaoqun, et al. Identification of tortoise shell and turtle shell by specific amplification technology [J]. Chinese Patent Medicine, 2018, 40(10): 2328-2333.

Table 2

According to Figure 1, it can be seen that the primers in the references did not achieve the specific identification of the turtle shell in this study, so the specific The opposite-sex primers were redesigned.

Based on the analysis of the Cytochrome Oxidase I sequence of the soft-shelled turtle in the Chinese Pharmacopoeia 2020 edition, the BioEdit software was used to perform a homologous comparison of the Cytochrome Oxidase I sequence of the soft-shelled turtle in the GeneBank database. The base sequences of the samples were imported into the Primer Premier 5 software for primer design.

After sequence comparison, it is found that the specific site of soft-shelled turtle is C and the others are A or T. After determining the SNP site, make the SNP site close to the 3' end of the forward primer, and adjust the primer score and GC by moving the position of the upstream primer. Content, and with the help of Primer Premier 5 software, further adjust the position of the reverse primer, and determine the best combination through the final primer score and product band score.

table 3

Considering the damage to the DNA base sequence after high temperature extraction, the length of the band of the turtle shell after specific PCR amplification is finally about 236bp.

(2) PCR amplification and electrophoresis

The reaction system for PCR amplification is 25 μL, including:

The reaction system was vortexed to mix and centrifuged briefly. Put the reaction system on the PCR instrument.

The amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, a total of 33 cycles; and final extension at 72°C for 5 min.

After the PCR amplification reaction is over, add 5 μL of 6×loading buffer to the reaction system, mix well, take 8 μL of the mixed product and spot on 1.5% (w/v) agarose gel, and electrophoresis at 150V for 25 minutes , developed by GelRed, and finally observed and recorded on the gel imager.

The results are shown in Figure 2. In Figure 2, M. DNA maker DL 1000, 1. Turtle shell control medicinal material, 2-16. Turtle shell medicinal material, 17-18. Vinegar turtle slices, 19-25. Turtle shell standard decoction, 26-27. Vinegar turtle shell standard decoction, 28-30. Turtle shell formula granules, 31-32. Vinegar soft-shelled turtle formula granules, 33-34. Tortoise shell medicinal material, 35-36. Horned soft-shelled turtle medicinal material, 37-38. Florida soft-shelled turtle medicinal material, 39. Shanrui soft-shelled soft-shelled turtle medicinal material, N. Blank (ddH ₂ O).

According to Figure 2, it can be known that at 200bp-300bp, the differences between the turtle shell medicinal material, vinegared turtle shell decoction pieces, turtle shell standard decoction, vinegar turtle shell standard decoction, turtle shell formula granules, and vinegar turtle shell formula granules were compared with the turtle shell control medicinal materials. There is a single band at the same location, while none of the other shams and blanks appear.

In summary, the present invention provides a pair of specific identification primers, and the specific identification primers are used to amplify the DNA of the Chinese medicine formula granules or other water extract products of the turtle shell. The identification of its decoction pieces can also be used for the identification of water extract products of turtle shell and its decoction pieces, such as traditional Chinese medicine standard decoction and traditional Chinese medicine formula granules, so that it can be distinguished from counterfeit water extract products and solve the problem of water extract It is difficult to identify the difficult problem after the product loses its shape. The specific identification primer pair is used for identification, which has good specificity and high accuracy of results, and provides guarantee for the drug safety and clinical efficacy of the water extract products of the turtle shell and its decoction pieces.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present application, and the description thereof is relatively specific and detailed, but should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the scope of protection of the patent application should be based on the appended claims.

Claims

A pair of specific identification primers for turtle shells and its decoction pieces, traditional Chinese medicine formula granules and other water extract products, characterized in that the pair of specific identification primers comprises a base sequence as shown in SEQ ID NO:1. Primer and base sequence are as the reverse primer shown in SEQ ID NO:2.
A specific identification kit for turtle shells and its decoction pieces, traditional Chinese medicine formula granules and other water extract products, characterized in that the kit includes the pair of specific identification primers as claimed in claim 1, and amplification reagents.
The specific identification kit of turtle shell and its decoction pieces, traditional Chinese medicine formula granules and other water extract products according to claim 2, wherein the amplification reagent comprises amplification buffer, dNTP, DNA polymerase and water; the amplification buffer is 10×PCR Buffer I, and the DNA polymerase is Taq enzyme.
A specific identification method for turtle shells, turtle shell decoction pieces, turtle shell traditional Chinese medicine formula granules or other turtle shell water extract products, characterized in that the specific identification method comprises the following steps:

extracting DNA from the sample to be tested; and,

Use the specific identification primer pair described in claim 1 or the specific identification kit described in claim 2 or 3 to amplify the DNA, separate the resulting amplification product, and analyze the sample to be tested according to the obtained separation product. for specific identification.
The specific identification method for turtle shells, turtle shell decoction pieces, turtle shell Chinese medicine formula granules or other turtle shell water extract products according to claim 4, characterized in that, according to the obtained separation product, the specificity of the sample to be tested is carried out Identification includes:

If the isolated product comprises a fragment of about 236bp, it means that the sample to be tested comprises turtle shells;

If the isolated product does not contain a fragment of about 236bp, it means that the sample to be tested contains fake turtle shells or does not contain turtle shells.
According to the specific identification method of turtle shells, turtle shell decoction pieces, turtle shell Chinese medicine formula granules or other turtle shell water extract products according to claim 4 or 5, it is characterized in that, said turtle shell counterfeit products are selected from turtle shell, horned One or more of softshell turtles, softshell turtles and softshell turtles.
According to the specific identification method of turtle shells, turtle shell decoction pieces, turtle shell Chinese medicine formula granules or other turtle shell water extract products according to any one of claims 4 to 6, it is characterized in that said other turtle shell water extracts The product contains the standard decoction of Biejia traditional Chinese medicine.
According to the specific identification method of turtle shells, turtle shell decoction pieces, turtle shell Chinese medicine formula granules or other turtle shell water extract products according to any one of claims 4 to 7, it is characterized in that the annealing temperature of the amplification is about 59℃～61℃.
The specific identification method for turtle shells, turtle shell decoction pieces, turtle shell traditional Chinese medicine formula granules or other turtle shell water extract products according to any one of claims 4 to 8, characterized in that the amplified reaction system comprises: About 2.5 μL containing Mg 2+ 10×PCR Buffer I, about 2.5mmol/L dNTP, about 10μmol/L forward primer, about 10μmol/L reverse primer, about 5U/μL Taq enzyme, about 30ng~50ng template DNA and about 19.2μL water.
The method for specific identification of turtle shells, turtle shell decoction pieces, turtle shell Chinese medicine formula granules or other turtle shell water extract products according to any one of claims 4 to 9, wherein the amplification reaction procedure includes: Pre-denaturation at about 95°C for about 5 minutes; denaturation at about 95°C for about 30 seconds, annealing at about 60°C for about 30 seconds, extension at about 72°C for about 30 seconds, a total of about 33 cycles; and finally extension at about 72°C for about 5 minutes.