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Nucleotide sequence and molecular probe for identifying dendrobium officinale and applications of molecular probe

A technology of nucleotide sequence and Dendrobium candidum, which is applied in the direction of microbial measurement/inspection, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems affecting the quality of commercial Dendrobium candidum, with short time to achieve, less sample consumption, The effect of simple method

Active Publication Date: 2014-06-04
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, it is very common for commercial Dendrobium officinale to be fake in the market, which seriously affects the quality of commercial Dendrobium officinale

Method used

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  • Nucleotide sequence and molecular probe for identifying dendrobium officinale and applications of molecular probe
  • Nucleotide sequence and molecular probe for identifying dendrobium officinale and applications of molecular probe
  • Nucleotide sequence and molecular probe for identifying dendrobium officinale and applications of molecular probe

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Experimental program
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Embodiment 1

[0026] Embodiment 1: Preparation of Dendrobium candidum specific nucleotide sequence

[0027] 1. Genomic DNA Extraction

[0028] In view of the high content of secondary products such as polysaccharides in Dendrobium, the following methods were adopted in the experiment:

[0029] (1) Take about 0.3g of fresh leaves and grind them into powder with a mortar. Quickly transfer the powder to a 1.5ml centrifuge tube, add 1ml of extraction buffer (200mmol / L Tris-HCl, 50ml / L EDTA, 250mmol / L NaCl, β-mercaptoethanol with a volume fraction of 2% ), after mixing, rest on ice for 15 minutes, and centrifuge at 8000 r / min for 10 minutes at 4°C. Remove the supernatant, if the supernatant is relatively viscous, centrifuge again according to the above method until the supernatant becomes clear, the purpose is to remove secondary metabolites such as polysaccharides.

[0030] (2) Add 500 μl 65°C preheated extraction buffer (3% CTAB, 100 mmol / L Tris-HCl (PH=8.0), 25 mM EDTA; volume fraction of ...

Embodiment 2

[0044] Example 2: Preparation of Dendrobium candidum-specific nucleotide molecular probe SHF / SHR, PCR amplification

[0045] On the basis of obtaining the specific nucleotide sequence of Dendrobium officinale, use Primer Primer5.0 software to design the nucleotide sequence of SHF / SHR (respectively shown in SEQ ID NO.2, SEQ ID NO.3), Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. Then use the designed and synthesized primers SHF / SHR to amplify and detect 38 Dendrobium samples of 36 species. The PCR amplification system is

[0046]

[0047] The reaction program was pre-denaturation at 94°C for 4 min; 35 cycles (denaturation at 94°C for 1 min, annealing at 65°C for 1 min, extension at 72°C for 2 min); and finally extension at 72°C for 10 min.

[0048] Use 1.5% agarose gel electrophoresis to detect the PCR results, and the electrophoresis picture is as follows: figure 2 shown, from figure 2 (In the figure, channels 1 to 38 are 38 samples of Dendrobium...

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Abstract

The invention relates to a nucleotide sequence and a molecular probe for identifying dendrobium officinale and applications of the molecular probe. The nucleotide sequence for identifying the dendrobium officinale is as shown in SEQ ID NO.1. An upstream nucleotide molecular probe SHF for identifying the dendrobium officinale is as shown in SEQ ID NO.2 and a downstream nucleotide molecular probe SHR is as shown in SEQ ID NO.3. The invention also provides the applications of the nucleotide molecular probe SHF / SHR in identification of the dendrobium officinale. In the applications of the nucleotide sequence and the molecular probe disclosed by the invention, few samples are used; the overall operation can be finished with use of few samples; the accuracy and the sensitivity are high; the SHF / SHR is a specific molecular probe for the dendrobium officinale, and a negative reaction is performed if the dendrobe is of another type; the method is simple; detection is carried out through a PCR (Polymerase Chain Reaction) technology and can be finished in a short time, particularly in half a day.

Description

technical field [0001] The invention belongs to the technical field of identifying dendrobium officinale by means of molecular biology methods, and relates to a nucleotide sequence for identifying dendrobium officinale, a molecular probe and an application thereof. Background technique [0002] Dendrobium officinale Kimura et Migo is a plant of the genus Dendrobium in the family Orchidaceae. It is mainly distributed in Yunnan, Guangxi, Zhejiang, Anhui, Fujian, Sichuan and other provinces in my country. Dendrobium officinale was first listed as medicinal in "Compendium of Materia Medica", and listed as top grade in "Shen Nong's Materia Medica". Shouwu, Shenshan Ganoderma lucidum, Cordyceps sinensis, etc. are listed as "China's Nine Immortal Herbs". Dendrobium candidum is the first among the "Nine Immortal Herbs". Human immunity, eliminate tumors, inhibit cancer and so on. Due to the fact that Dendrobium candidum is very rare, over-harvesting and habitat destruction are seri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 王慧中冯尚国何仁锋崔艺庆
Owner HANGZHOU NORMAL UNIVERSITY
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