Application of bulbus fritillariae cirrhosae ITS1 sequence fragment and detection method of fritillary bulb varieties

A technology of sequence fragments and detection methods, applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve problems such as failure to detect

Inactive Publication Date: 2019-04-12
康美华大基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above methods can only identify the presence or absence of Fritillaria sichuanensis, and fail to detect the specific components of Fritillaria sichuanensis (mainly Fritillaria sichuanensis and Fritillaria chinensis) in the mixture in processed traditional Chinese medicine preparations (such as Fritillaria sichuanensis powder). ), so it is necessary to develop a more rapid and accurate molecular biology method for the detection of Chuan Fritillaria and other specific ingredients in traditional Chinese medicine preparations

Method used

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  • Application of bulbus fritillariae cirrhosae ITS1 sequence fragment and detection method of fritillary bulb varieties
  • Application of bulbus fritillariae cirrhosae ITS1 sequence fragment and detection method of fritillary bulb varieties
  • Application of bulbus fritillariae cirrhosae ITS1 sequence fragment and detection method of fritillary bulb varieties

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The genome extraction of the known Fritillaria sichuanensis and the genome extraction of the samples were carried out separately: two samples were prepared, sample 1 was a mixture of Fritillaria sichuanensis and Fritillaria chinensis, and sample 2 was a mixture of Fritillaria sichuanensis and Fritillaria chinensis;

[0037] Reagents: Lysis solution (L1), DNA extraction solution (E1), buffer solution (W1), rinse solution (W2), DNA eluent (TE solution);

[0038] Proceed as follows:

[0039] 1) Take fresh tissue or dry weight tissue of Fritillaria, add liquid nitrogen to fully grind;

[0040] 2) Quickly transfer the ground powder to a centrifuge tube pre-installed with 65°C preheated lysate L1 (before the experiment, add mercaptoethanol to the preheated L1 to make the final concentration 0.1% by mass), and quickly After inverting and mixing, place the centrifuge tube in a 65°C water bath for 20 minutes, and invert the centrifuge tube during the water bath to mix the sampl...

Embodiment 2

[0051] The genome obtained in Example 1 is subjected to PCR amplification, and the amplification system is as follows:

[0052]

[0053] Dilute to 50 μL with nuclease-free water;

[0054] The PCR amplification reaction program was: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 32 cycles; final extension at 72°C for 10 min; storage at 4°C;

[0055] The primer pairs used were:

[0056] ITS1-F1: 5'-CGTAACAAGGTTTCCGTAGGTGAA-3'

[0057] ITS1-R1: 5'-GCTACGTTTCTTCATCGAT-3';

[0058] The ITS1 sequence fragment of the PCR product and the ITS1 sequence fragment of the sample to be tested were obtained.

Embodiment 3

[0060] The ITS1 sequence fragments obtained in Example 2 were purified separately, and the steps were as follows:

[0061] 1) Take out Ampure XP beads 30 minutes in advance and place them at room temperature, shake and mix well before use;

[0062] 2) Pipette 90 μL of Ampure XP beads (1.8 times) into the EP tube containing 50 μL of PCR product, mix well by blowing continuously with a pipette 10 times, and incubate at room temperature for 5 minutes;

[0063] 3) Instantaneous centrifugation, put the EP tube on the magnetic stand, let it stand for 2 minutes until the liquid is clear, suck it up with a pipette and discard the supernatant;

[0064] 4) Keep the EP tube fixed on the magnetic stand, add 200 μL of freshly prepared 80% ethanol by mass (to ensure that the ethanol has completely submerged the magnetic beads), and carefully discard the supernatant after standing at room temperature for 30 seconds;

[0065] 5) Repeat step 4) once, and try to blot up the liquid at the botto...

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Abstract

The present invention discloses an application of a bulbus fritillariae cirrhosae ITS1 sequence fragment and a detection method of fritillary bulb varieties. The detection method comprises the following steps: a PCR amplification step, an annealing reaction step, an enzymatic cleavage step, an electrophoresis step and an identification step: when a 310 bp fragment is shown in electrophoresis bands, a sample to be tested contains bulbus fritillariae cirrhosae; when 134 bp and 172 bp are shown in the electrophoresis bands, the sample to be tested contains thunberg fritillary bulbs; and when 142bp and 188 bp fragments are shown in the electrophoresis bands, the sample to be tested contains bulbus fritillariae ussuriensis. The bulbus fritillariae cirrhosae ITS1 sequence fragment is applied inthe detection of the fritillary bulb varieties and conducts variety identification of a fritillary bulb mixture containing the fritillary bulbs.

Description

technical field [0001] The invention relates to an application of ITS1 sequence fragment of Fritillaria sichuanensis and a detection method of Fritillaria species, belonging to the technical field of molecular biology detection. Background technique [0002] Fritillaria is a perennial herb of the Liliaceae family. As a good medicine for clearing away heat and nourishing the lungs, reducing phlegm and relieving cough, it has a long history of application. There are more than 20 species of Fritillaria in my country with medicinal value, mainly distributed in Sichuan, Xinjiang, Gansu, Zhejiang and other places. Due to the different origins, the medicinal value of Fritillaria fritillary strains varies to a certain extent, among which Fritillaria sichuanensis has the highest medicinal value. According to the 2015 edition of the "Chinese Pharmacopoeia", there are currently six sources of Fritillaria sichuanensis, namely Fritillaria sichuanensis, Fritillaria dark purple, Fritillar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2521/301C12Q2565/125
Inventor 胡悦许冬瑾韩丽娟卢玺峰
Owner 康美华大基因技术有限公司
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