WO2023134738A1 - Paire d'amorces d'identification spécifique pour trionycis carapax et morceaux de décoction, granules de distribution de la médecine traditionnelle chinoise et autres produits extraits de l'eau associés, utilisation de la paire d'amorces d'identification spécifique et procédé d'identification - Google Patents

Paire d'amorces d'identification spécifique pour trionycis carapax et morceaux de décoction, granules de distribution de la médecine traditionnelle chinoise et autres produits extraits de l'eau associés, utilisation de la paire d'amorces d'identification spécifique et procédé d'identification Download PDF

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WO2023134738A1
WO2023134738A1 PCT/CN2023/072028 CN2023072028W WO2023134738A1 WO 2023134738 A1 WO2023134738 A1 WO 2023134738A1 CN 2023072028 W CN2023072028 W CN 2023072028W WO 2023134738 A1 WO2023134738 A1 WO 2023134738A1
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WIPO (PCT)
Prior art keywords
turtle
turtle shell
specific identification
chinese medicine
water extract
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PCT/CN2023/072028
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English (en)
Chinese (zh)
Inventor
罗宇琴
李国卫
谭斯尹
刑菊玲
宋叶
魏梅
陈向东
孙冬梅
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广东一方制药有限公司
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Publication of WO2023134738A1 publication Critical patent/WO2023134738A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • 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.
  • 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.
  • 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
  • a pair of specific identification primers for turtle shells and its decoction pieces, traditional Chinese medicine formula granules and other water extract products 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 contains said pair of specific identification primers, and amplification reagents.
  • 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 comprising the following steps:
  • the specific identification of the sample to be tested according to the obtained separation product includes:
  • the isolated product comprises a 236bp fragment, it means that the sample to be tested comprises turtle shells,
  • 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.
  • the counterfeit turtle shell includes one or more of the turtle shell, horned turtle shell, Florida turtle shell and Shan Rui shell shell.
  • the other turtle shell aqueous extract products include the standard decoction of turtle shell traditional Chinese medicine.
  • the annealing temperature of the amplification is 59°C-61°C.
  • the amplification reaction system includes: 2.5 ⁇ L of 10 ⁇ PCR Buffer I containing Mg 2+ , 2.5 mmol/L dNTP, 10 ⁇ mol/L forward primer, 10 ⁇ mol/L reverse primer, 5 U/ ⁇ L Taq enzyme, 30ng ⁇ 50ng template DNA and 19.2 ⁇ L water.
  • 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.
  • 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
  • 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).
  • the first aspect is 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.
  • 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 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 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.
  • 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.
  • 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.
  • PCR there is still a technical gap in the use of PCR to identify water extracts of turtle shells and their decoction pieces.
  • 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.
  • the specific identification primers of the present application are also suitable for raw material identification of turtle shells/vinegar turtle shells.
  • 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.
  • 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.
  • 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.
  • 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:
  • the specific identification of the sample to be tested according to the obtained separation product includes:
  • the isolated product comprises a 236bp fragment, it means that the sample to be tested comprises turtle shells,
  • 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.
  • 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.
  • the other turtle shell aqueous extract products include the standard decoction of turtle shell traditional Chinese medicine.
  • the annealing temperature of the amplification is 59°C-61°C.
  • the amplification reaction system contains: 2.5 ⁇ L 10 ⁇ PCR Buffer I containing Mg 2+ , 2.5 mmol/L dNTP, 10 ⁇ mol/L forward primer, 10 ⁇ mol/L reverse primer, 5 U/ ⁇ L Taq Enzyme, 30ng ⁇ 50ng template DNA and 19.2 ⁇ L water.
  • 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.
  • the sample to be tested is a turtle shell-like sample or a turtle shell decoction piece-like sample
  • the DNA is extracted using the SDS-proteinase K method.
  • the sample to be tested is a water extract product
  • the DNA is extracted using the CTAB method
  • proteinase K is added to the CTAB extract used in the CTAB method.
  • 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.
  • the structure is destroyed due to heating, and the degree of DNA degradation is similar to that of ancient DNA samples.
  • the required amount of DNA can be extracted from the damaged DNA to be tested for subsequent amplification.
  • 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.
  • FIG. 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).
  • 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.
  • the specific site of soft-shelled turtle is C and the others are A or T.
  • 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.
  • the length of the band of the turtle shell after specific PCR amplification is finally about 236bp.
  • the reaction system for PCR amplification is 25 ⁇ L, 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.
  • 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).
  • 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.

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Abstract

La présente demande concerne une paire d'amorces d'identification spécifique pour Trionycis carapax et des morceaux de décoction, des granules de distribution de la médecine traditionnelle chinoise et d'autres produits extraits de l'eau de celle-ci, l'utilisation de la paire d'amorces d'identification spécifique et un procédé d'identification. La paire d'amorces d'identification spécifique comprend une amorce directe telle que représentée dans SEQ ID NO : 1, et une amorce inverse telle que représentée dans SEQ ID NO : 2.
PCT/CN2023/072028 2022-01-14 2023-01-13 Paire d'amorces d'identification spécifique pour trionycis carapax et morceaux de décoction, granules de distribution de la médecine traditionnelle chinoise et autres produits extraits de l'eau associés, utilisation de la paire d'amorces d'identification spécifique et procédé d'identification WO2023134738A1 (fr)

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CN202210044243.XA CN114350819B (zh) 2022-01-14 2022-01-14 鳖甲及其饮片、中药配方颗粒和其他水提物制品的特异性鉴别引物对及其应用、鉴别方法

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CN114350819B (zh) * 2022-01-14 2024-07-30 广东一方制药有限公司 鳖甲及其饮片、中药配方颗粒和其他水提物制品的特异性鉴别引物对及其应用、鉴别方法

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