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Primer combination for PCR (Polymerase Chain Reaction) identification of long-noded pit viper medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and identification method of primer combination

A combination technology of traditional Chinese medicine formula granules and primers, which is applied in biochemical equipment and methods, recombinant DNA technology, measurement/testing of microorganisms, etc., can solve the problems of DNA loss of form and difficulty in identification, and achieve the effect of overcoming the interference of excipients

Pending Publication Date: 2022-06-24
GUANGDONG YIFANG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide a combination of primers for PCR identification of Aggresis medicinal materials, standard decoctions and Chinese medicine formula granules, which has good specificity and can effectively solve the problem of difficult identification after DNA loses its form

Method used

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  • Primer combination for PCR (Polymerase Chain Reaction) identification of long-noded pit viper medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and identification method of primer combination
  • Primer combination for PCR (Polymerase Chain Reaction) identification of long-noded pit viper medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and identification method of primer combination
  • Primer combination for PCR (Polymerase Chain Reaction) identification of long-noded pit viper medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and identification method of primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Primer Design

[0074] Based on the NADH; Cytochrome Oxidase I; 12S rRNA; 16S rRNA; Cytochrome b sequence analysis of Chinese Pharmacopoeia 2020 edition, the Cytochrome Oxidase I sequences of common snake species in the GeneBank database were compared using BioEdit software. Yes, after proofreading, analyze the specific SNP site of Qi Snake, and import the base sequence containing the SNP site into Primer Premier 5 software for primer design.

[0075] After the sequence comparison, it was found that the specific site of the snake is T and the other is C or G. 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 upstream primer position. content, and with the help of PrimerPremier 5 software, the position of the reverse primer was further adjusted, and the optimal combination was determined by the final primer score and the product band score. In the design process, it is ...

Embodiment 2

[0078] Example 2 Establishment of identification method

[0079] (1) DNA extraction and concentration adjustment

[0080]Take 0.5 g of the dried sample, grind it into powder, put it in a 2 mL centrifuge tube, add 1.5 mL of CTAB precipitation solution preheated at 56 °C, 20 μL of proteinase K, mix well, heat it in a water bath at 56 °C for 60 min, and cool it to room temperature. Centrifuge at / min for 5 min, discard the supernatant, add 900 μL CTAB precipitation solution and 20 μL proteinase K, and operate in the same way. Add 900 μL of LCTAB extract and 10 μL of β-mercaptoethanol to the centrifuge tube in turn, mix well, heat in a water bath at 65°C for 120 min, centrifuge, and after cooling to room temperature, take the supernatant, add an equal volume of chloroform-isoamyl alcohol (24:1 ), shake for 3 min, mix well, centrifuge at 12000 r / min and 4°C for 10 min, take 750 μL of supernatant into a new 2 mL centrifuge tube, take the supernatant, and add an equal volume of chlo...

Embodiment 3

[0092] Embodiment 3 Identification method verification

[0093] 15 snake samples of known species were selected (specifically shown in Table 1).

[0094] The samples were identified using the identification method established in Example 2. Among them, in the PCR system, the amount of DNA template is 50ng.

[0095] Table 1 Sample Sheet

[0096]

[0097]

[0098] Identification results such as figure 1 As shown in the figure, it can be seen from the figure that there is a band of 188bp in Qishe medicinal materials, Qishe standard decoction (lyophilized powder), and Qishe Chinese medicine formula granules; while other samples did not get this band. This result shows that the accuracy rate of the identification method in the present invention reaches 100%.

[0099] Example 4 Methodological Validation - Annealing Temperature

[0100] (1) Take the cymbidium viper numbered 1 to 3, 7 to 9 in table 1, the round-spotted viper numbered 17 to 18, and the short-tailed viper numb...

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Abstract

The invention discloses a primer combination for PCR (Polymerase Chain Reaction) identification of long-noded pit viper medicinal materials, standard decoction and traditional Chinese medicine formula granules, the primer combination comprises a primer I and a primer II, the primer I is a DNA (Deoxyribose Nucleic Acid) molecule with a nucleotide sequence shown as SEQ ID NO: 1 or a DNA molecule which is obtained by substituting, deleting and / or adding one or more nucleotides to the sequence shown as SEQ ID NO: 1 and has the same function as the DNA molecule shown as SEQ ID NO: 1; and the primer II is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 2. The invention further discloses application of the primer combination and an identification method based on the primer combination. By implementing the method, long-noded pit viper medicinal materials, standard decoction and traditional Chinese medicine formula granules can be effectively identified.

Description

technical field [0001] The invention relates to the technical field of traditional Chinese medicine identification, in particular to a primer combination used for PCR identification of Qi Snake medicinal materials, standard decoction and traditional Chinese medicine formula particles, and an application and identification method thereof. Background technique [0002] There are more than 200 species of snakes in my country, of which more than 20 are common snakes, including Agkistrodonacutus, Zaocys dhumnades, Bungarus multicinctus, Bungarusfasciatus, Ptyas mucosus, round snake. Daboia russelii, Naja naja, Lycodon rufozonatus, Elaphe carinata, Ptyas korros, Gloydiusbrevicaudus, Elaphe taeniura, Elaphe moellendorffi, etc. Has clinical efficacy. Due to the similar shapes of various snakes, it is difficult to identify medicinal materials, especially after the internal organs are cut off during processing, they must be dried and blackened. After the standard decoction is further ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6806C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/6806C12Q1/686C12Q2527/125C12Q2521/537C12Q2565/125
Inventor 魏梅罗宇琴孙冬梅宋叶范耀耀黄上书索彩仙
Owner GUANGDONG YIFANG PHARMA
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