Levisticum officinale detection kit and detection method

A technology for detection reagents and detection methods, applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve the problems of less effective medicine than Angelica sinensis and short growth period, and achieve easy operation and good application foreground effect

Inactive Publication Date: 2015-09-30
四川医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Because the medicinal effect of Angelica sinensis is not as good as that of Angelica sinensis, and the pharmacology is also quite different, it cannot be used as Angelica sinensis. However, it has medicinal effects, is easy to cultivate, and has a short growth period. It is very necessary to use

Method used

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  • Levisticum officinale detection kit and detection method
  • Levisticum officinale detection kit and detection method
  • Levisticum officinale detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 RAPD technology amplifies the specific SCAR marker of Angelica sinensis

[0022] 1. Extraction of plant DNA by CTAB method

[0023] Take 0.2g of Angelica sinensis and put it in a 1.5mL EP tube, add liquid nitrogen for about half a minute and grind it into a fine powder with a mortar stick, then add 500μl of CTAB extraction buffer [CTAB2%, Tris-HCl (pH8.0) immediately 100mmol·L _1 , EDTA20mmol mmol·L _1 , NaCl 1.4mol L _1 , 0.4% β-mercaptoethanol], 60 ° C water bath for 1 hour, add an equal volume of chloroform-isoamyl alcohol (24:1) for extraction, gently invert and mix, centrifuge at 10,000 rpm for 10 minutes, and draw up For the supernatant, add 2 / 3 volume of pre-cooled isopropanol to the supernatant, mix it upside down gently; centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, pour off the supernatant carefully, and use 70% ethanol for precipitation Wash lightly once with absolute ethanol, discard the washing solution, keep the precipitat...

Embodiment 2

[0047] Example 2 Using the method of the present invention to specifically amplify the gene of Angelica sinensis

[0048] 1. Experimental method

[0049] (1) According to the Angelica sinensis-specific SCAR marker (sequence shown in SEQ ID NO.1) obtained in Example 1, a pair of primers were designed and synthesized.

[0050]

[0051] (2) PCR amplification

[0052] 1. Template preparation

[0053] Detection of different species: 18 different species or varieties of genomic DNA were selected as templates, which are: Gansu Minxian Angelica, Japanese Angelica, Sichuan Aba Angelica, Gansu Pingliang Angelica, Hubei Angelica, Sichuan Jiuzhai Angelica, European Angelica (7th Lanes), Yunnan Lijiang Angelica, Sichuan Mianyang Angelica, Ginkgo, Lychee, Longan, Qingguo, Honeysuckle, Gardenia, Ganoderma lucidum, Longli, Huangcao. They were diluted to 10ng / μl for later use.

[0054] Identify Japanese Angelica, European Angelica and Angelica: 1, 2 and 3 are Japanese Angelica, from Cho...

Embodiment 3

[0073] Example 3 Angelica detection kit and method of use of the present invention

[0074] 1. Kit composition

[0075] This kit contains:

[0076] CTAB extraction buffer (200ml);

[0077] 2×Taq Master Mix, wherein the primers can be selected from a pair of primers or multiple pairs of primers (500 μl) in the primers shown in SEQ ID NO.5-6, SEQ ID NO.7-8, and SEQ ID NO 9-10;

[0078] One tube (50 μl) of negative control template DNA (Japanese angelica or angelica DNA);

[0079] One tube (50 μl) of positive control template DNA (Angelica sinensis DNA);

[0080] Sterilized ddH 2 O (1500 μl).

[0081] (1) CTAB extraction buffer

[0082]

[0083] Adjust to pH 5.0 with HCL, add H 2 O to 100ml.

[0084] (2) Formula of PCR system 2×Taq Master Mix

[0085]

[0086] 2. Detection method

[0087] (1) PCR amplification

[0088] 1. CTAB extraction buffer is used to extract the DNA of the sample to be tested.

[0089] 2. Perform PCR amplification:

[0090] details as foll...

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PUM

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Abstract

The invention discloses a nucleotide sequence of levisticum officinale, and further discloses application of a reagent for detecting the nucleotide sequence in preparing a levisticum officinale detection reagent, and a levisticum officinale detection kit and a detection method. The detection kit and the detection method disclosed by the invention can be adopted to accurately and effectively identify levisticum officinale, and are good in specificity, short in operation time and good in application prospect.

Description

technical field [0001] The invention relates to a detection kit and detection method of Angelica sinensis. Background technique [0002] Angelica officinale (Levisticum officinale Koch), also known as Angelica sinensis, is a perennial herb belonging to the Umbelliferae family. In 1957, angelica was introduced from Bulgaria when it was in short supply, and it has been cultivated in my country's Hebei, Beijing, Shandong, Henan, Inner Mongolia, Liaoning, Shaanxi, Shanxi and Jiangsu. Angelica is used as medicine with dried roots, which was recorded in the German Pharmacopoeia early. It has a specific and turbid aroma, sweet in nature, slightly spicy, slightly sweet and numb tongue, and has the effects of diuresis, invigorating the stomach, eliminating phlegm, and treating gynecological diseases. , is a common counterfeit product of genuine angelica. [0003] Because the medicinal effect of Angelica sinensis is not as good as that of Angelica sinensis, and the pharmacology is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 傅俊江梅志强张春成竞梁魏春莉
Owner 四川医科大学
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