Molecular identification method of ground beeltle in ground beeltle medicinal material and Huoxue Zhitong Capsules

A technology for molecular identification, invigorating blood and relieving pain, applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems such as difficulty in obtaining the unique strips of S. The quality of the finished drug, easy to follow-up experiments, and highly specific effects

Active Publication Date: 2011-08-03
NANJING ZHONGSHAN PHARMA CO LTD
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Problems solved by technology

[0005] The inventors have also tried to use the random amplified polymorphism (RAPD) technique to identify the wood beetles in the Huoxuezhitong capsules. The experiment found that for the Huoxuezhitong capsules of different manufacturers and different batch numbers, the corresponding soil beetles could not be obtained. Negative control substance, because the genomes of the same medicinal material from different origins are different, which is reflected in the difference of amplified bands in the RAPD map, and it is difficult to obtain the unique bands of the ground beetle

Method used

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  • Molecular identification method of ground beeltle in ground beeltle medicinal material and Huoxue Zhitong Capsules
  • Molecular identification method of ground beeltle in ground beeltle medicinal material and Huoxue Zhitong Capsules
  • Molecular identification method of ground beeltle in ground beeltle medicinal material and Huoxue Zhitong Capsules

Examples

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Embodiment 1

[0054] The method for extracting the genome DNA of the wood beetle from the medicinal material of the wood beetle: the live wood beetle is killed by soaking in 95% ethanol for 3-5 minutes, and then dried or dried and crushed to 80 meshes. Take an appropriate amount (about 0.1g, grind and transfer it into a 2mL centrifuge tube to no more than 0.5 scale) ground beetle medicinal powder, put it into a mortar, add homogenization buffer and grind until it becomes a paste, transfer it to a 2mL centrifuge tube, add homogenization buffer Liquid to 1mL, mix well. Water bath at 58°C for 5 hours, and gently invert and mix every half hour during this period. After the water bath, cool to room temperature. Add an equal volume of saturated phenol: chloroform: isoamyl alcohol (25:24:1), mix by inversion slowly, centrifuge at 8000r / min for 10min, and transfer the supernatant to a new centrifuge tube. Repeat this step until there is no protein layer between the water layer and the phenol laye...

Embodiment 2

[0062] Get wild (PY) and cultured (DY) ground beetles and soak them in 95% ethanol for 3-5min to death, and dry or dry to obtain wild (PY) and cultured (DY) ground beetle medicinal materials respectively; get ground beetle medicinal materials according to the Pharmacopoeia method The Huoxue Zhitong Capsules were prepared, and the genomic DNA of the ground beetle was extracted according to the method in Example 1. Among them; wild wood beetle (live body), collected in Pingyi, Shandong; cultured wood beetle (live body), purchased from Danyang, Jiangsu.

[0063] Using 16Sra (SEQ ID NO.1) and 16Srb (SEQ ID NO.2) as primers and using the extracted genomic DNA of Wood Beetle as a template, 16S rRNA of Wood Beetle was amplified by PCR. The PCR amplification system is: total volume 25 μL, dNTPs (10 mmol / L) 2 μL, 10*Taq buffer (containing Mg 2+ ) 2.5 μL, Taq enzyme 0.6U, each primer (5 μmol / L) 1 μL, DNA template 1 μL, double distilled water to make up the volume, and finally add 10 μL...

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Abstract

The invention belongs to the identification field of the traditional Chinese medicinal compound preparation and discloses a molecular identification method of ground beeltle in the ground beeltle medicinal material and the Huoxue Zhitong Capsules. The method comprises the following steps: extracting and purifying the genomic DNA of ground beeltle; (2) perform polymerase chain reaction (PCR) amplification to the 16S rRNA of ground beeltle; and (3) performing electrophoresis assessment to the amplification product. By adopting the method, the genomic DNA of ground beeltle can be extracted successfully and the mitochondrion 16S rRNA fragment of ground beeltle can be amplified. Therefore, the authenticity, type and producing area of ground beeltle can be identified accurately and whether the capsules contain ground beeltle can be identified accurately.

Description

technical field [0001] The invention belongs to the identification field of traditional Chinese medicine compound preparations, and relates to a molecular identification method of the wood beetle medicinal material and the blood beetle relieving pain capsule. Background technique [0002] Many of the currently commercially available Chinese patent medicines contain traditional Chinese medicine powder raw materials. Due to the complex composition of powdered traditional Chinese medicines and many interfering factors, some traditional Chinese medicines are extremely difficult to detect by powder identification and chemical analysis. In recent years, traditional Chinese medicine preparations made by ultrafine grinding have destroyed the characteristics of cell tissue and increased the difficulty of inspection. Traditional identification techniques are helpless, which is not conducive to the identification and quality standardization of traditional Chinese medicine. However, tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 濮存海王云灵
Owner NANJING ZHONGSHAN PHARMA CO LTD
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