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PCR detection method of Dendrobium chrysotoxum by adoption of specific primers

A Dendrobium drumstick, specific technology, applied in biochemical equipment and methods, microbiological measurement/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome probe preparation, high operator requirements, and high cost, and achieve Good specificity, high sensitivity, and easy operation

Active Publication Date: 2012-10-17
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RFLP technology is stable and reproducible, but the preparation of probes is cumbersome; RAPD method can reveal the evolutionary relationship between relative species very well, but it is unstable and limited by experimental conditions; ISSR markers are fast, simple, and highly polymorphic. advantages, but its primers have poor versatility and amplification conditions require targeted design; AFLP technology perfectly combines RFLP and PCR technology, but its stability is poor, and it requires high operator requirements and costs a lot

Method used

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  • PCR detection method of Dendrobium chrysotoxum by adoption of specific primers
  • PCR detection method of Dendrobium chrysotoxum by adoption of specific primers
  • PCR detection method of Dendrobium chrysotoxum by adoption of specific primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 is used for detecting the synthesis of the PCR primer of dendrobium nodule

[0024] A total of 170 ITS sequences of Dendrobium genus Dendrobium, Dendrobium nobile, Dendrobium fringe, and Dendrobium were obtained from GenBank, and the above sequences were compared with the biological software MEGA 5.0 to find out all the mutation sites of Dendrobium different from other species. According to the position of the mutation site, primers of about 20 bases were designed on the upstream and downstream of the ITS sequence respectively; the designed primers were analyzed using the biological software DNAMAN, and the appropriate primer pair was finally determined. The upstream primer F 5'- CAAAGGATGGACGAACCCA-3' and downstream primer R5'-CGCAGCCAACGAGATGATT-3'.

[0025] Primer synthesis was completed by Nuosai Company.

Embodiment 2

[0026] Embodiment 2 Utilizes PCR primer to detect the specificity and sensitivity analysis of Dendrobium drumstick

[0027] 1.1 Sample source

[0028] The Dendrobium used in this example includes Dendrobium chrysalis, Dendrobium nobile, Dendrobium fringe, Dendrobium candidum, Dendrobium spp. Dendrobium sheathes, Dendrobium spp., and Dendrobium dendrobii were purchased from Ya'an in Sichuan, Ruili in Yunnan, Baise in Guangxi, Chishui in Guizhou, Simao in Yunnan, and Xishuangbanna in Yunnan.

[0029] 1.2 DNA extraction

[0030] Take about 30 mg of the above samples, add sterilized small steel balls, grind them into powder with a sample grinder, and use the kit method to extract DNA. The kit was purchased from TianGen Company, and the model was Plant Genomic DNA Extraction Kit (spin column type) 200preps.

[0031] 1.3 Serial dilution of sample DNA

[0032] The DNA concentration of the Dendrobium chrysalis sample extracted above was detected to be 20.7 ng / μl. The DNA samples ...

Embodiment 3

[0040]Example 3 Utilizes PCR primers to detect commercially available Dendrobium chrysanthemum samples

[0041] 1.1 Collect 16 different Dendrobium samples from the market, take about 30 mg of each sample, add sterilized small steel balls, grind them into powder with a sample mill, and use the kit method to extract DNA. The kit was purchased from TianGen Company, and the model was Plant Genomic DNA Extraction Kit (spin column type) 200preps.

[0042] 1.2 Use ITS universal primers ITS 5F: 5'-GGAAGTAAAAGTCGTAACAAGG-3' and ITS 4R: 5'-TCCTCCGCTTATTGATATGC-3' to perform PCR to detect the quality of each extracted DNA sample.

[0043] PCR reaction system:

[0044]

[0045] The PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 50°C for 1 min, 72°C for 1.55 min, a total of 30 cycles; 72°C for 7 min.

[0046] Set up a blank control and use an equal amount of ddH for the DNA template 2 O instead, prove that the quality of the DNA meets the detection requirements, and d...

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Abstract

The invention provides a PCR detection method of Dendrobium chrysotoxum by the adoption of specific primers. Based on ITS sequence of Dendrobium chrysotoxum, the specific primers F and R (as shown in Seq ID No.1 and 2) which can rapidly detect Dendrobium chrysotoxum are designed, and a PCR detection system is established on this basis so as to provide molecular identification basis for identification of the medicinal material Dendrobium chrysotoxum. According to the invention, Dendrobium chrysotoxum can be rapidly and accurately detected from a plurality of medicinal materials. A detection kit constructed according to the method is simple to operate, has good specificity and high sensitivity, can be used to realize detection of Dendrobium chrysotoxum's stem, leaf or other positions of the plant and medicinal materials, and is suitable for extensive popularization and application.

Description

technical field [0001] The invention relates to molecular biology detection of Dendrobium chrysotoxum, in particular to a method for detecting Dendrobium chrysotoxum by using specific primer PCR. Background technique [0002] Dendrobium, which has been recorded in "Shan Hai Jing", is listed as top grade in "The Classic", and is a precious Chinese medicinal material in my country. Sweet in nature, slightly cold, returns to the stomach and kidney channels, has the effects of nourishing the stomach and promoting body fluid, nourishing yin and clearing away heat. Dendrobium nobile Lindl., Dendrobium chrysotoxum Lindl., Dendrobium fimbriatum Hook. and their similar species in the same genus are recorded in "Chinese Pharmacopoeia" (2010 edition). fresh or dried stems. Modern pharmacological studies have shown that Dendrobium contains polysaccharides and other components, which can promote the digestive system, as well as anti-aging and immune regulation, anti-tumor, and cataract...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 陈士林黄林芳郑司浩
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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