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Identification method of PCR-restriction map for fritillary bulb and similar medicine material

An identification method, restriction endonuclease technology, applied in the direction of DNA preparation, recombinant DNA technology, fermentation, etc., can solve the problems of distinguishing Fritillaria fritillaria from counterfeit products, achieve reliable identification method, low toxicity, and not easy to cultivate Effect

Inactive Publication Date: 2005-11-16
CHINA PHARM UNIV
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Problems solved by technology

However, it is difficult to distinguish Chuan Fritillaria from counterfeit products with the usual identification methods such as morphology and microscopy. Therefore, finding a reliable method for the identification of Chuan Fritillaria is an important issue in the identification of Fritillaria.

Method used

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Embodiment Construction

[0013] 1. DNA extraction: proceed as usual, and adjust the DNA concentration of the test sample to 0.2-0.5 μg / μl with deionized water;

[0014] 2. Polymerase chain reaction:

[0015] (1) For polymerase chain reaction, 30 μl is used as a reference, and the dosages of various items are as follows:

[0016] 10×PCR buffer 3μl

[0017] MgCl 2 (25mM) 2.4μl

[0018] dNTP (each 10mM) 0.6μl

[0019] Primer P1, P2 (30pmol / μl) each 0.5μl

[0020] 1~2 μl DNA template for test product

[0021] TaqDNA polymerase 1U (0.2μl)

[0022] Deionized water make up to 30μl

[0023] (2) PCR reaction conditions: the reaction is carried out on a PCR instrument, and the reaction conditions are:

[0024] Pre-denature at 95°C for 3 minutes, and then carry out 30 cycles according to the following conditions:

[0025] Denaturation at 95°C for 20-40 seconds

[0026] Refolding at 53°C for 20-40 seconds

[0027] Extend at 72°C for 20-40 seconds

[0028] Keep at 72°C for 2 to 4 minutes to make up aft...

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Abstract

The present invention belongs to the field of Chinese medicine material quality identifying technology, and aims at providing one kind of PCR-RFLP method for identifying fritillary bulb and similar medicine material accurately. PCR amplification adopts the primer series including P1:5'-CGT AAC AAG GTT TCC GTA GGTGAA-3' and P2: 5' -GCT ACG TTC TTC ATC GAT-3'; the characteristic base sequence of fritillary bulb is 5'-TGCCCGCCCTGCCCGGGACCTCGC-3'; and the restriction endonuclease Sma I and its isoschizomer can identify and incise the sequence. The identification process includes: extracting medicine DNA, PCR amplification with the primers P1 and P2, digesting the PCR product with restriction endonuclease Sma I, agarose gel electrophoresis to obtain the analysis result, compared with fritillary bulb PCR-RFLP characteristic result to judge whether the sample is fritillary bulb.

Description

1. Technical field: [0001] The invention relates to the technical field of quality identification of germplasm of traditional Chinese medicinal materials. 2. Background technology: [0002] The traditional Chinese medicine Fritillaria sirrhosa is the dried bulb of Fritillaria cirrhosa D.Don, Fritillaria unibracteata Hsiao et K.C.Hsia, Fritillaria przewalskii Maxim, or Fritillaria delavayi Franch. It has the effects of clearing away heat and moistening the lungs, resolving phlegm and relieving cough. It is used for the treatment of dry cough due to lung heat, dry cough with less phlegm, overworked cough due to yin deficiency, and bloody phlegm. Due to its good curative effect, low toxicity, limited natural resources, and difficulty in artificial cultivation, it is the most valuable variety of Fritillaria medicinal materials, and the market price is very high. It can be seen in the market that small bulbs of other species of Fritillaria are used to pretend to be Chuan Fritill...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12P19/34C12Q1/68
Inventor 李萍王冲之
Owner CHINA PHARM UNIV
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