Molecular biological method for rapidly identifying trichosanthin medicinal material

A trichosanthin and molecular technology, applied in the field of molecular identification of trichosanthin and its analogs, can solve the problems of the origin and storage time of medicinal materials, poor repeatability of results, application limitations and the like

Inactive Publication Date: 2013-06-05
QINGDAO VLAND BIOTECH INC
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Problems solved by technology

However, there are disadvantages such as poor reproducibility of results and being easily affected by the place of origin and storage time of med...

Method used

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Embodiment Construction

[0005] Three single primers were designed according to the ITS sequence in the trichosanthin ribosomal RNA gene: TkS1-64F, Tks2-1 12F, Tks2-130R. The PCR reaction system is 25 μL, in which Tris-HCl (pH 9.0) 10mmol L -1 , KC1 50mmol·L -1 , Mg 2+ 1.5 mmol·L -1 , each dNTP 0.15 mmol·L -1 , Taq 1U (promega), Primer 0.15 μmol L -1 , template DNA 50 ng. Amplification was carried out on ABI9700. The amplification program was pre-denaturation at 95°C for 4 min, followed by 40 cycles: denaturation at 95°C for 30 s, annealing at 50°C for 45 s, extension at 72°C for 60 s, and extension at 72°C for 5 min after the cycle.

[0006] Among the three primers, TkS1-64F has a good amplification effect, showing that all TCS can be amplified, and most counterfeit products can be amplified, with 1-5 polymorphic bands, clear bands and good repeatability, which can be used for TCS Identification of medicinal materials. The identification bands of trichosanthum were determined to be 560, 960 bp...

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Abstract

The invention relates to a new molecular identification method for identifying trichosanthin and its similar products; adventurous innovation is made in two key links of primer design and annealing temperature. Primer design is performed on a known ITS of trichosanthin medicinal materials; the length is about 20 bp; and general primer design principles are followed. Meanwhile, the annealing temperature is screened and determined according to the number of polymorphism strips. The characteristic identification strips of trichosanthin are determined to be 560, 960 bp strips, and 1930, 1400, 839, 715 bp strips are considered as auxiliary identification strips of trichosanthin. The invention has the following advantages of: (1) simplicity and practicality, wherein although the difficulty for primer design is increased slightly, the specificity is greatly improved, an ideal identification primer can be obtained after design of 2-3 primers, and thus the problem of large screening of random primers is avoided; (2) stability and repeatability, wherein since the specificity and annealing temperature of the primer are improved, the amplification strips are generally only 1-5 strips, and certified products with different producing areas and different storage time have no effect on PCR results; (3) large provided information content, wherein both certified products and most fake products can be amplified simultaneously, and thus standard identification electrophoretograms of the certified products and fake products can be established to realize accurate identification of the certified products and fake products.

Description

technical field [0001] The invention relates to a new molecular identification method for identifying trichosanthemi and its analogues, belonging to the technical field of traditional Chinese medicines. Background technique [0002] There are 84 species and 8 varieties of Trichosanthes in the world, and 37 species and 6 varieties in China. There are many kinds of medicinal plants in this genus, 2 kinds are recorded in Pharmacopoeia 2005 edition, and 7 kinds are recorded in "Chinese Materia Medica", such as T. kirilowii Maxim. , its fruit (Gualou), root (Tianhua Fen) are commonly used in traditional Chinese medicine. The trichosanthin protein derived from trichosanthum pollen has been widely used in the induction of labor in early and middle trimesters, the treatment of ectopic pregnancy, hydatidiform mole and other diseases, and in recent years it has been used in the treatment of AIDS. The survey found that the sources of trichosanthes used in various places are complex,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 郝智慧钟英杰王艳玲
Owner QINGDAO VLAND BIOTECH INC
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