Molecular biological identification method of fritillaria taipaiensis

A technology of molecular biology and Taibai Fritillaria, applied in the field of traditional Chinese medicine identification, can solve the problems of complex splicing and assembly of sequencing data and difficulty in extracting sequencing templates

Inactive Publication Date: 2014-06-11
广州白云山潘高寿药业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are still some difficulties in the extraction of sequencing templates, and it is still complicated to assemble sequencing data when there are no seq

Method used

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  • Molecular biological identification method of fritillaria taipaiensis
  • Molecular biological identification method of fritillaria taipaiensis

Examples

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Embodiment 1

[0068] Example 1 Obtaining of Taibai Fritillaria Chloroplast Genome

[0069] (1) Extraction and purification of chloroplast genomic DNA from Taibai Fritillaria

[0070] Take fresh young leaves of Fritillaria taibai (fresh leaves of Fritillaria taibai were collected from Hongchiba, Wenfengdian, Wuxi County, Chongqing City (latitude: 31.4°, longitude: 109.6°), identified by the Institute of Medicinal Botany, Chinese Academy of Medical Sciences For Taibai Fritillaria.taipaiensis P.Y.Li,), adopt the improved sucrose density gradient centrifugation method to separate, extract and purify the chloroplast genome (cpDNA), the steps are as follows:

[0071] ①Take 100g of fresh young leaves, treat them in the dark at 4°C for 24 hours, wash them, remove the veins, tear them into pieces about 1cm2 in size, add 500mL of buffer A pre-cooled at 4°C, and place them in a homogenizer, so that the leaves are basically submerged. homogenate. Then filter once with 4 layers of gauze and once with ...

Embodiment 2

[0093] Example 2 Feasibility of identifying Taibai Fritillaria based on the complete sequence of the chloroplast genome

[0094] (1) Biological barcode identification of Taibai Fritillaria and Chuan Fritillaria based on single-gene sequences

[0095] Genomic DNA of medicinal materials was extracted according to methods known in the prior art, and species identification was performed using chloroplast trnh-psbA sequences. The primers used for the amplification of trnh-psbA region are:

[0096] Upstream primer: 5'-GTTATGCATGACGTAATGCTC-3'

[0097] Downstream primer: 5'-CGCGCATGGTGATTCACAATCC-3'.

[0098] PCR amplification conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 54°C for 1 min, extension at 72°C for 1.5 min, 30 cycles, and finally extension at 72°C for 7 min.

[0099] After sequencing, Fritillaria trnH-psbA sequence:

[0100] ACAATTTCCCTCTAGACCTAGCTGCTATTGAAGTTCCATCTACAAATGGATAAGACTTACATTCTGTCTTAGTGTATACGAATCCTTGATGGAGCAATA...

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Abstract

The invention discloses a molecular biological identification method of fritillaria taipaiensis. The method comprises the following steps: extracting chloroplast genome DNA (Desoxvribose Nucleic Acid) of a medicinal material to be identified; performing sequencing and sequence splicing to obtain a complete chloroplast genome sequence; comparing the sequence with the chloroplast genome sequence of fritillaria taipaiensis to realize rapid and accurate identification of fritillaria taipaiensis.

Description

technical field [0001] The invention belongs to the technical field of identification of traditional Chinese medicines, and specifically discloses a molecular biological identification method of Fritillaria taibai. Background technique [0002] Taibai Fritillaria, also known as Taibei, Jianbei, and Qinbei, is the dry bulb of Fritillaria L., a plant of the genus Fritillaria L. in the Liliaceae family Liliaceae. It is mainly used to treat dry cough due to lung heat, dry cough with less phlegm, and yin deficiency. Labor cough, expectoration with blood, are the varieties included in the 2010 edition of "Pharmacopoeia of the People's Republic of China". Both Taibai Fritillaria and Wabu Fritillaria are used as Chuan Fritillaria in Pharmacopoeia. In addition, Chuan Fritillaria, Suosha Fritillaria, Gansu Fritillaria and Dark Purple Fritillaria have 6 source plants of Chuan Fritillaria, but their appearance They are very similar, with only minor variations. Traditional classical tax...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q1/6895
Inventor 卢其福陈士林黄喜梅李西文刘薇黄林芳高丽周立
Owner 广州白云山潘高寿药业股份有限公司
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