Primer pair for identifyingtraditional Chinese medicine, namely, akebia stems, as well as application of primer pair

A primer pair, traditional Chinese medicine technology, applied in the field of molecular biology, can solve the problems of low accuracy, cumbersome methods, and high subjectivity, and achieve the effect of accurate identification

Active Publication Date: 2015-04-01
华润三九现代中药制药有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Different researchers used high-performance liquid chromatography, near-infrared spectroscopy, and ultraviolet spectroscopy to identify three different sources of Akebia (Li Rui, Fan Xialei, Zhou Xiaohua, etc. Guan Akebia, Chuan Akebia, and Sanye Akebia Identification of near-infrared diffuse reflectance spectroscopy, Chinese Journal of Pharmaceutical Industry, 2008, 42(12): 933; Xie Lisha, Tan Yuanfeng. Identification of three different genera of Akebia by ultraviolet spectroscopy, Shanghai Journal of Traditional Chinese Medicine, 2004, 38( 9):58), but these methods are cumbersome and not conducive to popularization
The detection methods based on molecular biology developed in recent years, such as DNAbarcoding and site-specific PCR, have overcome the shortcomings of traditional identification methods such as low accuracy and high subjectivity.
Molecular specific identification between Akebia, Chuanketong and Guankeke has not been reported yet

Method used

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  • Primer pair for identifyingtraditional Chinese medicine, namely, akebia stems, as well as application of primer pair
  • Primer pair for identifyingtraditional Chinese medicine, namely, akebia stems, as well as application of primer pair
  • Primer pair for identifyingtraditional Chinese medicine, namely, akebia stems, as well as application of primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the preparation and use method of the kit for identification of traditional Chinese medicine Akebia

[0042] 1. Design and synthesis of primer pairs for identification of Chinese medicine Akebia

[0043] In this example, the general primer trnL-trnF (trnL: 5'-GGAAATCGGTAGACGCTACG-3'trnF: 5'-ATTTGAACTGGTGACACGAG-3') was selected to amplify the three traditional Chinese medicine samples of Mutong, Chuanmutong and Guanmutong in Table 1. Sequencing, and then performing homologous alignment on the obtained sequences, and designing a pair of specific primers F1 and F2 according to the variable region, as a pair of specific primers for identifying Akebia. The sequence is as follows:

[0044] Primer F1: 5'-AGTTGGCTGCGTTGCGT-3' (SEQ ID NO: 1);

[0045] Primer F2: 5'-AGTGGAGTGAATGAGAAACTTAGC-3' (SEQ ID NO: 2).

[0046] 2. Assembly of kits for identification of Chinese medicine Akebia

[0047] After the primers F1 and F2 designed and synthesized in step 1 are pac...

Embodiment 2

[0060] Embodiment 2, using the kit prepared in Example 1 to identify the traditional Chinese medicine Akebia

[0061] Samples to be tested: 5 kinds of samples shown in Table 1, Akebia, Sanye Akebia, Bai Ketong, Chuan Akebia, and Guan Ke.

[0062] 1. Extract genomic DNA from the sample to be tested

[0063] Select about 0.03 g of dry medicinal material without mildew, put it in a pulverizer, grind it, and pass it through a 40-mesh sieve. Transfer the powder to a 2.0 mL microcentrifuge tube, add 900 μL of sterilized CTAB extract (recipe: 2% (2g / 100ml) CTAB, 100mmol / L Tris-HCl pH=8.0, 20mmol / L EDTA, 1.4mol / L NaCl), 0.02g PVP 40000, 10μL β-mercaptoethanol, fully vortexed and mixed, 65 ℃ water bath for 1.5h-2h, during which, shake gently 2-3 times. After completion, take it out and cool to room temperature, add 900 μL of chloroform-isoamyl alcohol (volume ratio 24:1), shake and mix well, and centrifuge at 12000 g for 10 min. Take the supernatant, add an equal volume of chlorofo...

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Abstract

The invention discloses a primer pair for identifying a traditional Chinese medicine, namely, akebia stems, as well as an application of the primer pair. Theprimer pair for identifyingor assisting in identifying the traditional Chinese medicine, namely, the akebia stems, specifically consists of two single-stranded DNAmolecules represented in Sequence 1 and Sequence 2 in a sequence table. Tests prove that the traditional Chinese medicine, namely, the akebia stems, armand clematis stems and manchurian dutchmanspipe stems are identified quickly and accurately by the aid of the primer pair with a rapid PCR (polymerase chain reaction) method and a fluorescence staining method, authenticity identification results are detected with an introduced fluorescent dye method, and the technical support is provided for field application of medicine molecule identification.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a pair of primers for identifying the traditional Chinese medicine Akebia and its application. Background technique [0002] The traditional Chinese medicine Akebia is the stem of Akebia quinata (Thunb.) Decne, Akebia trifoliate (Thunb.) Koidz. or Akebia trifoliate (Thunb.) Koidz.var.australis (Diels) Rehd. rattan. Diuretic for treating stranguria, clearing heart and irritability, stimulating menstrual flow and breastfeeding. It can cure stranguria syndrome, edema, upset urine, sore mouth and tongue, etc. [0003] According to literature research, the authentic product of Akebia used in ancient times is Akebia family. The Qing Dynasty "Plant Names and Facts" proposed that Ranunculaceae Akebia, that is, Chuan Akebia. There is no record of "Guan Mutong" in the herbal medicines of the past dynasties. In 1954, Ren Ren'an found through investigation that my country's commercial ake...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/166C12Q2563/107
Inventor 黄璐琦袁媛崔占虎蒋超
Owner 华润三九现代中药制药有限公司
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