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A standard gene sequence of kudzu dna barcode and its application

A barcode and gene technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, biochemical equipment and methods, etc., can solve the problems of unsuitable industrialization and popularization of detection, high requirements for samples to be measured, and complex result judgment methods, etc. problems, to achieve the effects of shortening the identification time, simple result determination, and low cost

Active Publication Date: 2021-02-23
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many deficiencies in these traditional detection or identification methods. For example, the material components of Pueraria species are relatively similar, especially for closely related species, chromatographic analysis is often difficult to distinguish well, while RAPD molecular marker analysis, SRAP molecular Although methods such as marker analysis and ITS sequence analysis can distinguish the closely related species of Pueraria genus, these methods have high requirements for samples to be measured. When the content of Pueraria mirifica in the sample is low, false Positive or False Negative Questions
In addition, RAPD molecular marker analysis and SRAP molecular marker analysis need to compare complex electrophoretic patterns, and ITS sequence analysis requires sequence composition analysis. The results of these methods are complicated to determine and have high requirements for experimental equipment. They are not suitable for industrialization , popularized testing

Method used

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  • A standard gene sequence of kudzu dna barcode and its application
  • A standard gene sequence of kudzu dna barcode and its application
  • A standard gene sequence of kudzu dna barcode and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Analysis of the amplification of kudzu DNA barcode standard detection gene sequence and its PCR primers in different species

[0038] 1. Experimental materials

[0039] 1.1 Plant material

[0040] Eight different varieties of Pueraria lobata were collected from eight different provinces in my country (from Hunan, Shanxi, Zhejiang, Hubei, Jiangxi, Sichuan, Guizhou, Anhui). The other 20 different species of materials are: Pueraria thomsonii, Pueraria omeiensis, soybean (Glycine max), long cowpea (Vigna unguicμLata), pea (Pisum sativum), broad bean (Vicia faba), Mung beans (Vigna radiata), peanuts (Arachis hypogaea), Arabidopsis thaliana (Arabidopsis thaliana), tobacco (Nicotiana tabacum), rice (Oryza sativa), corn (Zea mays), barley (Coix chinensis), radish (Raphanus sativus), barley (Hordeum v μLgare), wheat (Triticum aestivum), potato (Solanum tuberosum), tomato (Lycopersicon esc μLentum), rapeseed (Brassica napus), cotton (Gossypium spp).

[0041] 1.2 Enzy...

Embodiment 2

[0069] Example 2 Kudzu DNA Barcode Standard Detection Gene Sequence and Detection Sensitivity Analysis of PCR Primers

[0070] 1. extract the kudzu genome DNA, the extraction method is the same as the method in Example 1.

[0071] 2. Dilute the kudzu genomic DNA with water to 20ng / μL, 2ng / μL, 0.2ng / μL, 0.02ng / μL, 0.002ng / μL, and use the primer combination F1 / R1 of the present invention as primers for PCR amplification , the method of PCR amplification is identical with the method in embodiment 1

[0072] 3. Results analysis, such as Figure 4 As shown, the results show that there are still clearly visible bands when the DNA concentration is as low as 0.02ng / μL, indicating that the detection method disclosed by the present invention can detect samples with a genomic DNA content as low as 0.02ng / μL, and has high sensitivity.

Embodiment 3

[0073] Example 3 Pueraria mirifica DNA barcode standard detection gene sequence and its PCR primers are used for the detection of Pueraria components in the commercially available kudzu powder Chinese medicine

[0074] 1. Experimental materials

[0075] 1.1 Plant material

[0076] Instant Pueraria Mirifica Powder (commercially available), Pueraria Mirifica Powder (Tong Ren Tang brand), Pueraria Mirifica Herbal Pieces (commercially available), Pueraria Mirifica Meal Replacement Powder (commercially available), Pueraria Mirifica Powder (Yuguiyuan Brand), Pueraria Mirifica Ding ( commercially available)

[0077] 1.2 Enzymes and Reagents

[0078] Molecular biology reagents, TAKARA Taq DNA polymerase, 10×PCR buffer, dNTP Mixture (10mM), MgCl 2 purchased from Dalian Bao Biological Company. PCR primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0079] 1.3 Experimental Instruments

[0080] PCR instrument: DNA Engine Dyad Peltier Thermal Cyc...

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Abstract

The present invention relates to a standard gene sequence of a kudzu DNA barcode and its application. The standard detection gene sequence of the barcode is shown in SEQ ID NO.1. The genome of Pueraria mirifica was amplified by polymerase chain reaction. According to the size judgment result of the amplified product of the tested sample, if a band of 226bp can be specifically amplified, it can be judged that the tested sample contains kudzu components. . The invention provides a method for specifically detecting kudzu components, which is helpful for the identification of kudzu components, prevents adulteration and ensures the health of consumers. Compared with the traditional morphological identification method, the gene sequence obtained by the invention is beneficial to realize the molecular identification of kudzu, and can effectively shorten the identification time of kudzu.

Description

technical field [0001] The invention relates to the field of species identification, in particular to a kudzu species-specific gene sequence and a primer composition thereof. Background technique [0002] Pueraria lobata (scientific name: Pueraria lobata, English name: kudzu) is a perennial deciduous vine of the genus Pueraria. Except for Xinjiang, Qinghai and Tibet, it is distributed several times throughout the country. Pueraria mirifica is a sun plant, and it often grows in patches on the edge of the forest on the sunny slope of 100-2000 meters above the poster or in the bushes by the river. The whole body of Pueraria mirifica is treasure, and its root, stem, leaf, flower, fruit (seed) etc. all have higher edible value, medicinal value and economic value. Pueraria lobata "Compendium of Materia Medica" records: Pueraria lobata has a sweet, flat, non-toxic smell. It has the effects of clearing heat and detoxifying, relieving spasm and relieving pain, promoting yang and r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11C12Q1/686
CPCC12Q1/686C12Q1/6895C12Q2565/125
Inventor 张丽郑丽平刘秤利覃瑞刘虹李刚方莹
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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