Method for identifying cordate houttuynia and herb of Chinese gymnotheca

A technology of Houttuynia cordata and 100-step revival, which can be used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc.

Pending Publication Date: 2016-05-25
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, for dried or processed samples, traditional methods are difficult to accurately identify
However, identification by

Method used

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  • Method for identifying cordate houttuynia and herb of Chinese gymnotheca
  • Method for identifying cordate houttuynia and herb of Chinese gymnotheca
  • Method for identifying cordate houttuynia and herb of Chinese gymnotheca

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1. Design of multiplex PCR primers for identifying Houttuynia cordata and Baibu Huanhun

[0060] 1. Genomic DNA extraction

[0061] Genomic DNA of Houttuynia cordata Thunb and Gymnothe cachinensis Decne were extracted by alkaline lysis method. The specific steps of the above-mentioned alkaline lysis method for extracting genomic DNA are as follows:

[0062] 1) Take 10-25mg of dry material (Houttuynia cordata or Baibu Huanhun), crush it, and put it into a 2.0ml centrifuge tube.

[0063] 2) Add 200 μL of alkaline lysis extract solution (NaOH 0.5M, PVP 1% and Triton×1001%), and vortex well to mix.

[0064] 3) Add 800 μL of neutralizing solution (Tris-HCl 0.1M, pH=8.0), shake and mix well, centrifuge at 12000 rpm for 1 min, discard the precipitate, and take the supernatant.

[0065]4) Take 500 μL of supernatant to a new 2.0ml centrifuge tube, add 500 μL of neutralizing solution (Tris-HCl 0.1M, pH=8.0), invert and mix well, centrifuge at 12000 rpm for 1 min, and t...

Embodiment 2

[0079] Embodiment 2, the method for distinguishing Houttuynia cordata and Baibu Huanhun

[0080] 1. Specific PCR to identify samples of Houttuynia cordata and Houttuyniae reincarnation

[0081] (1) Extraction of genomic DNA

[0082] Genomic DNA of Houttuynia cordata Thunb and Gymnothecachinensis Decne in Table 1 were extracted by alkaline lysis method.

[0083] Table 1. Information of experimental materials

[0084]

[0085] (2) PCR amplification

[0086] Using the genomic DNA in step (1) as a template and using primers HcF and HgR to perform PCR amplification to obtain PCR products.

[0087] PCR reaction system: a total volume of 20 μL, including 1 U of TopTaq DNA polymerase (QIAGEN, 200201), 2.0 μL of 10×buffer, 20 nmol of dNTP, 2 μL of 2 μM primers (HcF and HgR), and 20 μL of sterilized distilled water.

[0088] PCR reaction program: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 1 min, 72°C extension for 7 mi...

Embodiment 3

[0128] Embodiment 3, the annealing temperature sensitivity detection of multiplex PCR primer

[0129] 1. Genomic DNA extraction

[0130] Genomic DNA of Houttuynia cordata Thunb and Gymnothecachinensis Decne in Table 1 were extracted by alkaline lysis method.

[0131] 2. Multiplex PCR amplification

[0132] Respectively using the genomic DNA in step 1 as a template, using the method of multiplex PCR in step 2 and 3 in Example 2 to quickly identify the Houttuynia cordata and the Houttuynia reincarnation samples, and using different annealing temperatures (50°C, 50.5°C, 51.6°C , 53.2° C., 55.1° C., 56.7° C., 57.6° C., 58° C.) were subjected to PCR amplification respectively, and the remaining reaction conditions were unchanged to obtain PCR products respectively.

[0133] (3) Electrophoresis

[0134] PCR products were electrophoresed on 1.2% agarose gel. The results showed that under the condition of annealing temperature of 50-58°C (50°C, 50.5°C, 51.6°C, 53.2°C, 55.1°C, 56.7...

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Abstract

The invention discloses a method for identifying cordate houttuynia and herb of Chinese gymnotheca. The method comprises the steps that a primer 1, a primer 2 and a primer 3 are used for carrying out PCR amplification on a sample to be detected, and the size of a PCR amplification product is detected through electrophoresis; if the PCR amplification product contains fragments of 185 bp and does not contain fragments of 389 bp, the sample to be detected is cordate houttuynia; if the PCR amplification product contains the fragments of 389 bp and does not contain the fragments of 185 bp, the sample to be detected is herb of Chinese gymnotheca; if the PCR amplification product contains the fragments of 185 bp and the fragments of 389 bp, the sample to be detected is a mixed sample of cordate houttuynia and herb of Chinese gymnotheca. Whether cordate houttuynia and herb of Chinese gymnotheca are mixed or not can be identified based on a built multiple PCR method through a single PCR, a thought is provided for later mutual identification relevant to cordate houttuynia and herb of Chinese gymnotheca, and the method has practical application value in preventing mixing and misuse of medicinal materials.

Description

technical field [0001] The invention relates to a method for distinguishing Houttuynia cordata and Baibu Huanhun, belonging to the field of biotechnology. Background technique [0002] Houttuynia cordata Thunb, a traditional Chinese medicine, is the fresh whole herb or dry aerial part of Houttuynia cordata Thunb. The traditional Chinese medicine Baibu Huanhun is the whole grass or leaves of the same family plant Gymnothecachinensis Decne. The efficacy and indications of the two are different, but because of the same distribution, often mixed growth, and similar shapes, it is easy to misuse and misuse; Houttuynia cordata and Baibu Huanhun can be identified from traditional methods such as the morphology and microscopic characteristics of the original plant. for identification. But for dried or processed samples, traditional methods are difficult to accurately identify. The use of chemical composition analysis for identification is very cumbersome and complicated, and it is...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/16
Inventor 黄璐琦袁媛魏艺聪赵玉洋
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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