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Primer set, kit and method for identifying the type of Desmodium spp.

A technique for identifying the type of Desmodium spp., a kit for identifying the type of Desmodium spp., and a primer set for identifying the type of D. Versatility, high accuracy, and high stability

Active Publication Date: 2018-03-30
WUHAN KANGLE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the current identification of Desmodium sativa still needs to be improved

Method used

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  • Primer set, kit and method for identifying the type of Desmodium spp.
  • Primer set, kit and method for identifying the type of Desmodium spp.
  • Primer set, kit and method for identifying the type of Desmodium spp.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Materials, Instruments and Reagents

[0060] 1. Sample collection of Desmodium spp.

[0061] The 15 samples used for the experiment were collected in Guangxi and Guangdong provinces in July and August 2014. The collected fresh leaves were wrapped with filter paper locally and stored in ziplock bags with color-changing silica gel at room temperature. for a week. See Table 1 for information on the source information of 15 copies of S. glabras.

[0062] Table 1 Sources of Desmodium spp.

[0063] Sample serial number

place of origin

Cultivated / wild

A

Kuangling Village, Zhongyong Township, Lingui District, Guilin City

cultivation

B

Sanpo Village, Siding Town, Rong'an County, Liuzhou City

cultivation

C

Dumei Village, Xindi Town, Cangwu County, Wuzhou City

half wild

D

Jilin Village, Qiaogong Township, Xingbin District, Laibin City

half wild

E

Dongling Village, Wutong Town, Lingui County,...

Embodiment 2

[0068] Solution configuration

[0069] 1. 50×TAE solution

[0070] Accurately weigh 242g of Tris base, 57.1ml of glacial acetic acid, and 100ml of 0.5mol / L EDTA, adjust the pH to 8.0 with NaOH, and dilute to 1L with water, and set aside.

[0071] Electrophoresis buffer: Dilute 50×TAE solution 50 times with pure water to obtain 1×TAE working solution.

[0072] 2. DNA extraction buffer

[0073]2% (W / V) CTAB, 3% (W / V) PVP, 1mol / L Tris-Hcl (pH=8.0) 50mL, 0.5mol / L EDTA-Na 2 (pH=8.0) 20mL, NaCl 41g, water to 500ml. After sterilization, add 1000 μL of β-mercaptoethanol.

[0074] 3. Nucleic acid dye working solution

[0075] After diluting Sybra Green I 100 times with 1×TAE working solution, add 2 times the volume of 6×loadingbuffer, and mix well to obtain the working solution of nucleic acid dye.

Embodiment 3

[0077] Extraction of Genomic DNA from Desmodium spp.

[0078] The collected fresh Desmodium broadleaf leaves were absorbed and dried by color-changing silica gel, and then the surface of the leaves was washed with absolute ethanol. After the absolute ethanol was evaporated, they were cut into fine pieces with scissors. After adding liquid nitrogen, they were quickly ground into powder with a ball mill.

[0079] Quickly add the ground leaf powder (about 30 mg) to 800 μL of DNA extraction buffer (pre-added RNase 50 μg) preheated to 65 °C in a water bath at 65 °C for 25 minutes, and turn the EP tube upside down several times every 5 minutes to prevent the leaf powder from agglomerating. Piece. Take out the EP tube, put it on ice, add an equal volume of Tris-saturated phenol: chloroform: isoamyl alcohol (25:24:1), shake well, put it in a centrifuge, centrifuge at 10000rpm at 20°C for 10min, take 600μL Serum. Add an equal volume of chloroform:isoamyl alcohol (24:1) to the superna...

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Abstract

The invention provides a primer group, a kit and a method for identifying types of desmodium styracifolium. The primer group consists of a first primer, a second primer, a third primer, a fourth primer, a fifth primer, a sixth primer and a seventh primer, wherein the nucleotide sequences of the first primer, the second primer, the third primer, the fourth primer, the fifth primer, the sixth primer and the seventh primer are respectively shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7. The primer group has one or more of the following advantages: the universality is strong, the accuracy is high, the objectivity is strong and the stability is high.

Description

technical field [0001] The present invention relates to the field of biology. Specifically, the present invention relates to a primer set for identifying the type of Desmodium spp., a kit for identifying the type of Desmodium spp., and a method for identifying the type of Desmodium spp. Background technique [0002] Guangshencao, the name of traditional Chinese medicine, is the dry aboveground part of the leguminous plant Guangshencao. Its effects are diuresis and jaundice dispelling, inducing diuresis to relieve stranguria, mainly treating jaundice, red urine, hot stranguria, stone stranguria, astringent urination, edema and oliguria. Distributed in Fujian, Hunan, Guangxi and Guangdong provinces. [0003] However, the current identification of Desmodium sativa still needs to be improved. Contents of the invention [0004] The present invention aims to solve at least one of the technical problems existing in the prior art. For this reason, the object of the present inv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 王学海许勇李莉娥杨仲文冯芸尹海龙杨婷黄璐余通曹儒宾谢长
Owner WUHAN KANGLE PHARMA
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