Primer for multi-PCR (Polymerase Chain Reaction) recognition of bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and recognition method of primer

A technology of traditional Chinese medicine formula granules, money white snake, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc. , Overcome the effect of the interference of excipients

Pending Publication Date: 2022-02-15
GUANGDONG YIFANG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, standard decoctions and traditional Chinese medicine formula granules often contain a certain proportion of auxiliary materials, which makes it difficult to apply the existing PCR method.
Furthermore, during the preparation of standard decoctions and traditional Chinese medicine formula granules, heating and extraction are often required, resulting in DNA degradation, making it difficult to retain DNA fragments exceeding 200 bp, resulting in low detection accuracy of existing methods
In addition, the existing PCR method often can only realize the identification of a single species of snake, and the efficiency is low

Method used

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  • Primer for multi-PCR (Polymerase Chain Reaction) recognition of bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and recognition method of primer
  • Primer for multi-PCR (Polymerase Chain Reaction) recognition of bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and recognition method of primer
  • Primer for multi-PCR (Polymerase Chain Reaction) recognition of bungarus multicinctus medicinal material, standard decoction and traditional Chinese medicine formula granules as well as application and recognition method of primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Primer Design

[0087]Based on the NADH; Cytochrome Oxidase I; 12SrRNA; 16S rRNA; Cytochrome b sequence analysis of the Chinese Pharmacopoeia 2020 edition and common snake species, the homology comparison of the Cytochrome Oxidase I sequences of common snake species in the GeneBank database was performed using BioEdit software Yes, after proofreading, analyze the specific SNP site of Snake chrysalis, import the base sequence containing the SNP site into the Primer Premier 5 software, and design primers.

[0088] After sequence comparison, it was found that the specific site of the golden snake is T and the others are C. 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 content by moving the position of the upstream primer , 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 sco...

Embodiment 2

[0096] Embodiment 2 identification method is established

[0097] (1) DNA extraction and concentration adjustment

[0098] Take 0.5 g of 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 in a water bath at 56 °C for 60 min, cool to room temperature, and use Centrifuge at 10000r / min for 5min, discard the supernatant, then add 900μL CTAB precipitation solution, 20μL proteinase K, and operate in the same way. Add 900 μL CTAB extract and 10 μL β-mercaptoethanol to the centrifuge tube successively, mix well, heat in a water bath at 65°C for 120 min, centrifuge, and take the supernatant after cooling to room temperature; add an equal volume of chloroform-isoamyl alcohol (24: 1), shake for 3 minutes, mix well; then centrifuge at 12000r / min, 4°C for 10 minutes, take 750 μL of the supernatant and add it to a new 2mL centrifuge tube, add an equal volume of chloroform-...

Embodiment 3

[0110] Embodiment 3 identification method verification

[0111] All kinds of samples of white snake, red chain snake and golden krait were taken (as shown in Table 1).

[0112] The identification method established in Example 2 was used to identify the samples. Wherein, in the PCR system, the amount of DNA template is 50ng.

[0113] Table 1 Sample sheet

[0114]

[0115]

[0116] Identification results such as figure 1 As shown, it can be seen from the figure that the medicinal materials of white snakes, standard decoction (freeze-dried powder) of snakes, traditional Chinese medicine formula granules of snakes, and the mixture containing snakes medicinal materials, standard decoction containing snakes There is a band of 160bp in all the mixtures of snakes; while other snake samples do not get this band. It can be seen from the figure that there is a band of 207bp in the red chain snake medicinal material, the red chain snake standard decoction (freeze-dried powder), ...

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Abstract

The invention discloses a primer for multi-PCR (Polymerase Chain Reaction) recognition of bungarus multicinctus medicinal materials, standard decoction and traditional Chinese medicine formula granules. The primer comprises a first primer pair, a second primer pair and a third primer pair, wherein the sequence of an upstream primer of the first primer pair is as shown in SEQ ID NO: 1, and the sequence of a downstream primer of the first primer pair is as shown in SEQ ID NO: 2; the sequence of an upstream primer of the second primer pair is as shown in SEQ ID NO: 3, and the sequence of a downstream primer of the second primer pair is as shown in SEQ ID NO: 4; the sequence of an upstream primer of the third primer pair is as shown in SEQ ID NO: 5, and the sequence of a downstream primer of the third primer pair is as shown in SEQ ID NO: 6. The invention further discloses application of the primer and an recognition method based on the primer. By implementing the method, bungarus multicinctus, dinodon rufozonatum and krait can be effectively recognized at the same time.

Description

technical field [0001] The invention relates to the technical field of identification of traditional Chinese medicines, in particular to a primer for multiple PCR identification of Snake chrysalis medicinal materials, standard decoctions and traditional Chinese medicine formula granules and its application and identification method. Background technique [0002] There are more than 200 species of snakes in my country, of which more than 20 species are common snakes, including Agkistrodonacutus, Zaocys dhumnades, Bungarus multicinctus, Bungarus fasciatus, Ptyas mucosus, Daboia russelii, Naja naja, Lycodon rufozonatus, Elaphe carinata, Ptyas korros, Gloydiusbrevicaudus, Elaphe taeniura, Elaphe moellendorffi, etc. Most of them are not Has clinical efficacy. Due to the similar shape 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. The pattern features and color...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/6895C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 罗宇琴范耀耀魏梅孙冬梅陈向东黄上书宋叶索彩仙
Owner GUANGDONG YIFANG PHARMA
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