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Method of acquiring rDNA ITS2 sequence for identification of dendrobium candidum

A Tiepifengdou and sequence technology, which is applied in the field of identification of medicinal materials source varieties, can solve the problems of affecting DNA extraction efficiency, sample DNA degradation, low amplification efficiency, etc., and achieve the effects of easy control, high accuracy and simple operation.

Active Publication Date: 2017-02-15
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this technology has the following problems in the identification of Tiepi Fengdou: (1) During the process of processing Dendrobium candidum into Tiepi Fengdou, high-temperature heating is often carried out for processing, and the time is long, which causes serious degradation of sample DNA and affects DNA. Extraction efficiency; (2) Universal primer ITS2F / 3R has low amplification efficiency in Tiepi Fengdou ITS2 sequence amplification

Method used

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  • Method of acquiring rDNA ITS2 sequence for identification of dendrobium candidum
  • Method of acquiring rDNA ITS2 sequence for identification of dendrobium candidum
  • Method of acquiring rDNA ITS2 sequence for identification of dendrobium candidum

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Identification of Example 1 Tiepi Fengdou

[0023] 1. Experimental materials

[0024] In this case, a total of 69 samples of Tiepi Fengdou were collected, and the specific information is shown in Table 1.

[0025] Table 1 Tiepi Fengdou sample information

[0026]

[0027]

[0028]

[0029] 2. Experimental equipment

[0030] Desktop high-speed centrifuge, PTC-100 gene amplification instrument, electrophoresis instrument, ultraviolet gel imaging analysis system, MM400 ball mill (Germany Retsch).

[0031] 3. DNA extraction

[0032] Take 20-30 mg of dried Tiepi Fengdou medicinal materials, chop them up and crush them with MM400 ball mill, add 500-1000 μL of nuclear separation solution, mix well, centrifuge at 7000 r / min for 5 minutes, discard the supernatant, add 500-1000 μL of nuclear separation solution, repeat This step is repeated 3-5 times until the supernatant is not viscous. The remaining steps were carried out according to the standard operating method...

Embodiment 2

[0050] The identification of embodiment 2 Dendrobium officinale base plant

[0051] Basically the same as Example 1, except that the original plant of Dendrobium candidum was used as the sample, DNA was extracted according to the above method, and the ITS2 sequence was amplified. As a result, the target sequence was also successfully obtained and the identification was completed.

Embodiment 3

[0052] The identification of embodiment 3 dendrobium officinale seed seedlings

[0053] Basically the same as Example 1, the difference is that Dendrobium officinale seeds and seedlings are used as samples, DNA is extracted according to the above method, and the ITS2 sequence is amplified. As a result, the target sequence can also be successfully obtained and the identification is completed.

[0054]

[0055]

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Abstract

The invention discloses a method of acquiring an rDNA ITS2 sequence for identification of dendrobium candidum, provides a standard sequence for identification of dendrobium candidum, and can effectively solve the molecular identification problem of dendrobium candidum medicinal materials. The specific steps include: (1) designing two pairs of primers to conduct segmented PCR amplification on an ITS2 sequence; (2) sequencing the amplification results, and connecting the results together according to overlapping regions, thus obtaining an ITS2 sequence; and (3) performing comparison with the standard sequence SEQ ID No.1, and if the sequence similarity is greater than or equal to 99%, identifying the sample as Dendrobium officinale. The method provided by the invention can realize rapid and accurate identification of dendrobium candidum herbs, original plants, seeds and seedlings, etc.

Description

technical field [0001] The invention relates to the technical field of identification of source species of traditional Chinese medicinal materials, in particular to a method for quickly identifying Tiepi Fengdou by using a ribosomal DNA (rDNA) ITS2 fragment and an application thereof. Background technique [0002] Dendrobium officinale Kimura et Migo (the Latin name Dendrobium catenatum Lindley in the English version of "Flora of China" eflora) is a rare and precious medicinal plant of Orchidaceae, and has the reputation of "the first of the nine Chinese immortal grasses". Dendrobium officinale has strict requirements on the growth environment. Its natural reproductive capacity is low, its growth is slow, and after long-term excavation, its natural resources are on the verge of exhaustion. It is listed as a national key protected medicinal material. Tiepi Fengdou is processed from fresh strips of Dendrobium candidum and has been recognized as a high-end health product. At p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10
CPCC12Q1/6806C12Q1/6895C12Q2600/13C12Q2531/113
Inventor 姚辉马双姣陈士林宋经元杨兵勋杨培周红
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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