Indel-marked primers of Fritillaria taibai and its application

A technology for labeling primers and Taibai Fritillaria, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of poor morphological standardization of cultivated products, increased similarity of Fritillaria, and increased difficulty in identification, etc. problems, to achieve the effect of low cost, few operation steps, and short detection cycle

Active Publication Date: 2022-04-29
SOUTHWEST JIAOTONG UNIV
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0005] The identification of Taibai Fritillaria mainly relies on the traditional trait identification method. This method mainly observes and compares the characteristics of the bulb color, navel point shape, scale leaf number, cohesion tightness, top opening and closing, length and smell of the sample. It needs to be enriched. identification experience, and the standardization of cultivar morphology is poor
Coupled with human intervention in cultivation, the similarity of different Fritillaria is increasing, and the difficulty of identification is increasing. Generally, it is necessary to combine other analysis and identification techniques for identification.
Chemical methods such as high-performance liquid chromatography and liquid chromatography-mass spectrometry consume a lot of materials and require large-scale instrument support, making it difficult to popularize. Therefore, it is urgent to develop more convenient and rapid detection methods to meet the monitoring needs of the medicinal material market

Method used

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  • Indel-marked primers of Fritillaria taibai and its application
  • Indel-marked primers of Fritillaria taibai and its application
  • Indel-marked primers of Fritillaria taibai and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: The experiment and results of taking Taibai Fritillaria base plant and medicinal materials as samples

[0050] 1) DNA extraction of Taibai Fritillaria samples

[0051] Take 1 sample of Fritillaria Taipaiensis P.Y.Li, take about 50-60 mg each of Fritillaria Taipaiensis P.Y.Li leaves and dried bulb medicinal materials, wash with 75% alcohol and sterile ultrapure water, dry in the air, add liquid nitrogen and grind into For the ultrafine powder, use the Plant Genomic DNA Extraction Kit (DP305) of Beijing Tiangen Biochemical Co., Ltd. to extract the genomic DNA of the sample according to the instructions. The extracted DNA was detected using a Bio-Tek microplate reader, and sterilized ultrapure water was used as a blank control to detect the concentration of DNA.

[0052] 2) PCR reaction system and reaction parameters

[0053] The PCR reaction system is: 12.5 μL of 2×Master Mix (Beijing Qingke Biotechnology Co., Ltd.), 1.0 μL each of the upstream and downstream...

Embodiment 2

[0063] Embodiment 2: Experiments and results of other kinds of Fritillaria samples

[0064] 1) Genomic DNA extraction of other types of Fritillaria samples

[0065] Basically the same as Example 1, the difference is that the present embodiment collects totally 13 kinds of different Fritillaria base species (Table 3), including: dark purple Fritillaria (Fritillaria unibracteata), Taibai Fritillaria (Fritillaria taipaiensis), curly leaf Fritillaria cirrhosa, Fritillaria delavayi, Fritillaria wabuensis, Fritillaria przewalskii, Fritillaria cirrhosa var.ecirrhosa Franch, Fritillaria cirrhosa var. There are 13 species of Fritillaria mellea), Fritillaria sinica, Fritillaria thunbergii, Fritillariaussuriensis, Fritillaria hupehensis Hsiao et K.C.Hsia, and Fritillaria walujewii Regel 26 samples were subjected to genomic DNA extraction. The extracted genomic DNA was directly used in subsequent PCR experiments to detect the specificity of the PCR reaction.

[0066] Table 3 Samples of...

Embodiment 3

[0074] Operation steps of the application embodiment of the present invention:

[0075] 1. Take Taibai Fritillaria (including basic plants and medicinal materials) and other samples of Fritillaria 50-70mg;

[0076] 2. Add liquid nitrogen to grind into fine powder, and use the plant genome DNA extraction kit (DP305) to complete the genome DNA extraction;

[0077] 3. Use the BioTek microplate reader to determine the DNA concentration;

[0078] 4. Take 12.5 μL of DNA 2×Master Mix (Beijing Qingke Biotechnology Co., Ltd.), 1.0 μL each of the Indel marker primer upstream and downstream primers (both at a concentration of 10 μM) in step 2, and 2.0 μL of the DNA template. Bacterial ultrapure water was added to make up the volume to a total volume of 25 μL.

[0079] 5. Use Thermo Fisher 96-Well Thermal Cycler PCR instrument to perform PCR;

[0080] The reaction parameters are 95°C pre-denaturation for 10min, 1 cycle, (95°C denaturation 30S, 58°C annealing, 30S, 72°C extension, 50S, ...

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Abstract

The invention provides an Indel marker primer, method and application for quickly identifying Fritillaria taibai, and belongs to the technical field of identification of source varieties of Chinese medicinal materials. The present invention finds for the first time that the PCR method based on the Indel marker primer can specifically identify Taibai Fritillaria, and only Taibai Fritillaria shows positive results at the 302bp position, while other Fritillaria species show negative results. It can be used for the specific molecular identification of the original plants and medicinal materials of Taibai Fritillaria. The present invention has the following advantages: 1. good specificity, only Fritillaria Taibai shows a positive result at the 302bp position; 2. high sensitivity, the minimum detection concentration (DNA) of PCR is 0.239ng / μL; 3. few steps and short detection cycle: The operation time is only 2‑3 hours; ④ Good reproducibility: more than 10 biological repetitions have been carried out, and the reproducibility reaches 100%.

Description

technical field [0001] The invention belongs to the technical field of identification of source varieties of Chinese medicinal materials, and discloses an InDel marker primer of Taibai Fritillaria and a PCR method established by using the marker primers, which can accurately distinguish Taibai Fritillaria from other basic Fritillaria species, and effectively ensure the The original accuracy and quality safety. [0002] technical background [0003] The determination of biological species of Chinese medicinal materials derived from plants and animals is called the base. If there are two or more basic sources recorded under the standard source of Chinese medicinal materials, it is called multi-genetic Chinese medicinal materials. Although multi-element origin alleviates the market vacancy of traditional Chinese medicinal materials, the mixed use of multi-element origin seriously affects the effectiveness, safety and quality controllability of clinical curative effect of tradit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/156
Inventor 廖海周嘉裕张田王威威蒋瑞平
Owner SOUTHWEST JIAOTONG UNIV
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