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Method for detecting whether Araliaceae plant components exist in sample and whether sample is adulterate

A technology for the detection of samples and plant components, applied in biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc.

Inactive Publication Date: 2016-06-01
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Araliaceae plants are dicotyledonous plants, and Araliaceae plants that can be used for medicine include ginseng, Panax notoginseng, American ginseng, etc. At present, traditional morphological methods are mainly used for identification of Araliaceae plants, which rely on the experience of identifying personnel and are highly subjective , so an accurate and effective identification method is needed

Method used

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  • Method for detecting whether Araliaceae plant components exist in sample and whether sample is adulterate
  • Method for detecting whether Araliaceae plant components exist in sample and whether sample is adulterate
  • Method for detecting whether Araliaceae plant components exist in sample and whether sample is adulterate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Embodiment 1SEQIDNO:1 and 2 to the PCR amplification of notoginseng, ginseng, American ginseng

[0084] (1) Take 30 mg of Panax notoginseng, Panax ginseng and Panax ginseng powder respectively, and extract Panax notoginseng, Panax ginseng, Total DNA of American ginseng, specifically:

[0085] (a) adding liquid nitrogen to fully grind;

[0086] (b) Quickly transfer the ground powder to a centrifuge tube pre-filled with 700 μL of 65°C preheated buffer GP1 (add mercaptoethanol to the preheated GP1 before the experiment to make the final concentration 0.1%), and quickly invert to mix After homogenization, place the centrifuge tube in a 65°C water bath for 20 minutes, and invert the centrifuge tube during the water bath to mix the sample several times;

[0087] (c) Add 700 μL of phenol / chloroform (v / v1:1), mix thoroughly, and centrifuge at 12000 rpm (~13400 × g) for 5 min;

[0088] (d) Carefully transfer the upper phase of the aqueous layer obtained in the previous step i...

Embodiment 2

[0099] The comparison of embodiment 2SEQIDNO:1 and 2 and ITS2 universal primer

[0100] Taking the multi-flavored plant-derived medicinal materials in Dieda Huoxue Powder as an example, compare the PCR amplification results of SEQ ID NO: 1 and 2 with the ITS2 universal primer. The herbal medicinal materials in Dieda Huoxue Powder include safflower, angelica, rhizoma drynaria, Dipsacus, rhubarb and notoginseng medicinal materials, etc. Weigh 30 mg of each medicinal material powder and ginseng powder respectively, according to the step (1) in Example 1 Under the same conditions, extract the total DNA of each medicinal material; for the total DNA of each medicinal material, use the ITS2 universal primer and SEQ ID NO: 1 and 2 respectively, according to the step (2) in Example 1, carry out under the same conditions Real-time fluorescent PCR amplification, wherein, each sample is tested twice, and the sequence of the ITS2 universal primer is:

[0101] ITS2-3R: GACGCTTTCTCCAGACTACA...

Embodiment 3

[0106] Embodiment 3 Utilize SEQIDNO:1 and 2 to identify whether adulteration in the notoginseng sample to be detected

[0107] Take by weighing the very fine powder of 30mg Radix Notoginseng, Radix Ginseng, American Ginseng respectively, by the step (1) in the embodiment 1, extract the total DNA of Radix Notoginseng, Radix Radix Radix Ginseng, American Ginseng respectively under the same conditions; Using SEQIDNO: 1 and 2 as primers, according to the step (2) in Example 1, carry out real-time fluorescent quantitative amplification under the same conditions, and test the high-resolution melting curve of the amplification result obtained, the test condition is 95 ° C denaturation , renatured at 40°C for 30s, kept at 65°C for 1s, slowly increased the temperature from 65°C to 95°C at a rate of 4.4°C / s until the DNA double strands were completely opened, collected fluorescence during the heating process, and tested each sample twice. The results are as follows Figure 5A and Figu...

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Abstract

The invention provides a method for detecting whether Araliaceae plant components exist in a sample and whether the sample is adulterate. The method for detecting whether Araliaceae plant components exist in a sample comprises the following steps: extracting total DNA (deoxyribonucleic acid) from a sample to be detected; by using the extracted total DNA as a template, carrying out PCR (polymerase chain reaction) amplification by using a primer pair, wherein the primer pair comprises SEQ ID NO:1 and SEQ ID NO:2; and carrying out analytical judgment on the PCR amplification product to identify whether Araliaceae plant components exist in the detected sample. The SEQ ID NO:1 and SEQ ID NO:2 are designed according to the ITS2 cistron sequence on an eucaryote rDNA (complementary deoxyribonucleic acid). Compared with the ITS2 universal sequence, the SEQ ID NO:1 and SEQ ID NO:2 can be used for carrying out amplification on the ITS 2 segment sequence of Araliaceae plants and can not be used for amplifying plant drugs of other families. The method can be used for detecting whether Araliaceae plant components exist in the sample, especially ginseng, pseudo-ginseng and / or American ginseng components.

Description

technical field [0001] The invention relates to a method for detecting whether there are Araliaceae plant components in a sample and whether it is adulterated, in particular to a method for detecting whether a sample of Panax notoginseng to be inspected is adulterated. Background technique [0002] Araliaceae plants are dicotyledonous plants, and Araliaceae plants that can be used for medicine include ginseng, Panax notoginseng, American ginseng, etc. At present, traditional morphological methods are mainly used for identification of Araliaceae plants, which rely on the experience of identifying personnel and are highly subjective , so an accurate and effective identification method is needed. [0003] During the production and processing of mixtures containing Araliaceae plant components, such as medicinal materials, or traditional Chinese medicine preparations using Araliaceae plants as medicine, part of the DNA can usually be retained in it, therefore, the Araliaceae plan...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/142C12Q2600/158C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 王菲菲张聿梅戴忠马双成
Owner NAT INST FOR FOOD & DRUG CONTROL
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