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Kit and method for identifying pholidota chinensis

A kit, the technology of Shixiantao, is used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Accurate effect

Active Publication Date: 2021-11-16
SOUTHWEST MEDICAL UNIVERISTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most of the methods for identifying Shixiantao are visual observation or biochemical reagent identification, but the accuracy is difficult to guarantee

Method used

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  • Kit and method for identifying pholidota chinensis
  • Kit and method for identifying pholidota chinensis
  • Kit and method for identifying pholidota chinensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 RAPD technology amplifies Shixiantao specific SCAR markers

[0028] 1. Extraction of plant DNA by CTAB method

[0029] Take 0.2g of Dendrobium or Shixiantao sample and place it in a 1.5mL EP tube. After adding liquid nitrogen for half a minute, grind it into a fine powder with a mortar, and immediately add 500 μL of CTAB extraction buffer [CTAB2%, Tris-HCl (pH8 .0)100mmol . L -1 , EDTA20mmolmmol . 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 preci...

Embodiment 2

[0054] Embodiment 2 uses the method of the present invention to specifically amplify the gene of Shixiantao

[0055] 1. Experimental method

[0056] (1) According to the specific SCAR marker (sequence shown in SEQ ID NO.1) obtained in Example 1, a pair of primers shown in Table 2 were designed and synthesized.

[0057] Table 2 SCAR sequence, PCR conditions and product size

[0058]

[0059] (2) PCR amplification

[0060] 1. Template preparation

[0061] Detection of different species: select 20 different regions of Dendrobium, Dendrobium or other medicinal plant genomic DNA as templates, channels 1-20 respectively represent Dendrobium bee-waisted, Dendrobium chinensis, Dendrobium longsu, Dendrobium black hair, Dendrobium rose, Dendrobium Xiantao, Copper Dendrobium, Dendrobium, Dendrobium officinale, Dendrobium nobile, wolfberry, lychee, ginkgo, honeysuckle, mint, yellow grass, angelica, longan, gardenia, red ganoderma. Diluted to 10ng / μL respectively for use.

[0062] ...

Embodiment 3

[0079] Embodiment 3 Shixiantao detection kit and method of use of the present invention

[0080] 1. Kit composition

[0081] This kit contains:

[0082] CTAB extraction buffer (200mL);

[0083] 2×Taq Master Mix, wherein the primers are the primer pair shown in A6-33 in Table 2 (ie the primer pair shown in SEQ ID NO.5 and SEQ ID NO.6; 500 μL);

[0084] One tube of negative control template DNA (DNA of Dendrobium candidum or Dendrobium nobile) (50 μL);

[0085] One tube (50 μL) of positive control template DNA (Shixiantao DNA);

[0086] Sterilized ddH 2 O (1500 μL).

[0087] (1) CTAB extraction buffer formula

[0088] CTAB extraction buffer (100mL) formula is as follows:

[0089]

[0090] Adjust to pH 5.0 with HCl, add H 2 0 to 100 mL.

[0091] (2) Formula of PCR system 2×Taq Master Mix

[0092] The formula of 2×Taq Master Mix (500μL) is as follows:

[0093]

[0094] 2. Detection method

[0095] (1) PCR amplification

[0096] 1. CTAB extraction buffer is used ...

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Abstract

The invention discloses a kit and a method for identifying pholidota chinensis, and belongs to the field of gene detection. The kit comprises a reagent for detecting the sequence as shown in SEQ ID NO. 1. It is found for the first time that the nucleotide sequence shown in SEQ ID NO.1 is a pholidota chinensis specific sequence, whether a to-be-detected sample is pholidota chinensis or not can be accurately identified by detecting the sequence, pholidota chinensis, dendrobe and other varieties of medicinal plants are distinguished, and guidance is provided for reasonable use of medicinal materials. The kit can accurately and effectively identify pholidota chinensis and has strong specificity. The identification method is simple and convenient to operate, short in time consumption and high in test accuracy, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a kit and method for identifying shixiantao. Background technique [0002] Dendrobium (scientific name: Dendrobium nobile Lindl), also known as Immortal Dendrobium Orchid Rhyme, Immortal Grass, Resurrection Grass, Ziying Xianzhu, Chlorophytum and so on. The stem is erect, fleshy and thick, slightly flattened cylindrical, 10-60 cm long and 1.3 cm thick. Medicinal plant, sweet and slightly salty in nature and flavor, cold, and returns to the stomach, kidney, and lung meridian. Tonify stomach and promote body fluid, nourish yin and clear away heat. It is used for yin deficiency and body fluid deficiency, dry mouth, polydipsia, retching due to lack of food, asthenia heat after illness, and blurred vision. Dendrobium is a commonly used traditional Chinese medicine. It was first recorded in "Shen Nong's Materia Medica" and listed as top grade. This product is sweet in nat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/156C12Q2531/113C12Q2565/125Y02A50/30
Inventor 梅志强王恺付俊江李春红
Owner SOUTHWEST MEDICAL UNIVERISTY
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