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Primer pair for identifying radish seeds and application of primer pair

A primer pair and identification technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of strong subjectivity, cumbersome fluorescent staining methods, unfavorable standardization, etc.

Inactive Publication Date: 2015-07-08
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] To sum up, different researchers have used microscopic, physical and chemical methods and protein electrophoresis methods to identify Radix Seed medicinal materials, but the traditional methods of microscopic and physical and chemical methods are highly subjective, which is not conducive to standardization and standardization.
Protein electrophoresis technology takes a long time to test and requires electrophoresis to complete. Therefore, compared with fluorescent staining methods, it is cumbersome and is not conducive to the application and promotion of on-site rapid identification.

Method used

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  • Primer pair for identifying radish seeds and application of primer pair
  • Primer pair for identifying radish seeds and application of primer pair
  • Primer pair for identifying radish seeds and application of primer pair

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Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1, the preparation and the use method that are used for identifying the kit of radish seed

[0049] 1. Design and synthesis of primer pairs for identifying radish seeds

[0050] The general primer ITS (1F: 5'-TCCGTAGGTGAACCTGCGG-3'; 4R: 5'-TCCTCCGCTTATTGATATGC-3') was selected for the combination of radish seed, perilla seed, white mustard seed, dodder seed, green beetle seed, sand garden seed, rapeseed, acute seed, The samples of mallow seeds were amplified and sequenced, and then the obtained sequences were compared with the radish sequences downloaded from GenBank (GenBank accession numbers: AF128105, AF128104, AY746462, AY563100, AY662290, FJ980407), and a pair was designed according to the variation region. Specific primers F1, F2, the sequence is as follows:

[0051] F1: 5'-ATGCCTTCCGATTCCGTGGTTATTT-3' (SEQ ID NO: 1);

[0052] F2: 5'-ATGGGGGGATGACGATTTGTGAC-3' (SEQ ID NO: 2).

[0053] 2. Assembly of kits for identifying radish seeds

[0054] After t...

Embodiment 2

[0067] Embodiment 2, the kit identification that adopts embodiment 1 preparation Radix Semen Radix

[0068] Samples to be tested: 9 samples of Chinese herbal medicines shown in Table 1.

[0069] 1. Extract genomic DNA from the sample to be tested

[0070] Select about 0.03 g of dry medicinal material without mildew, put it in a pulverizer, grind it, and pass it through a 40-mesh sieve. Transfer the powder to a 2.0 mL microcentrifuge tube, add 900 μL of sterilized CTAB extract (recipe: 2% (2g / 100ml) CTAB, 100mmol / L Tris-HCl pH=8.0, 20mmol / L EDTA, 1.4mol / L NaCl), 0.02g PVP 40000, 10μL β-mercaptoethanol, fully vortexed and mixed, 65 ℃ water bath for 1.5h-2h, during which, shake gently 2-3 times. After completion, take it out and cool to room temperature, add 900 μL of chloroform-isoamyl alcohol (volume ratio 24:1), shake and mix well, and centrifuge at 12000 g for 10 min. Take the supernatant, add an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1), shake and mi...

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Abstract

The invention discloses a primer pair for identifying radish seeds and an application of the primer pair. The primer pair for identifying or helping to identify the radish seeds is specifically composed of two single-chain DNA molecules as shown in a sequence 1 and a sequence 2 in the sequence table. Experiments prove that the primers are adopted to realize quick and accurate identification of radish seeds, Chinese dodder seeds, perilla fruits, white mustard seeds, feather cockscomb seeds, flastem milkvetch seeds, seeds of garden balsam, chingma abutilon seeds, rapeseeds and the like are identified by use of a fast PCR method and a fluorescent staining method; and a fluorescent dye method is introduced to detect the true and false identification results, and technical support is provided for the field application of molecular identification of medicinal materials.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, relates to the field of identification of traditional Chinese medicines and medicinal materials, in particular to a pair of primers for identifying radish seeds and an application thereof. Background technique [0002] According to a record in the 2010 edition of "Chinese Pharmacopoeia", the source of radish is the dried mature seeds of the radish Raphanus sativus L., a cruciferous plant. Laifuzi is a commonly used traditional Chinese medicine in clinical practice. It has the effect of eliminating indigestion and distension, reducing qi and reducing phlegm. In recent years, radish seeds are often confused with the seeds of the same family plants, Brassica sinensis, rapeseed, and dodder or southern dodder in the commodity market. In order to ensure the quality and clinical efficacy of radish in the medicinal material market, it is necessary to Establish a more rapid and accurate method ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 黄璐琦袁媛崔占虎蒋超
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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