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Primer pair used for identifying peucedanum praeruptorum and application thereof

A primer pair and pedicel technology, applied in DNA preparation, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of unfavorable realization of fast, on-site detection, and long time consumption

Inactive Publication Date: 2015-09-09
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The DNA barcoding technology used by Xiong Yongxing et al. to identify Qianhu and its counterfeit products (Xiong Yongxing, Wu Lan, Liu Yimei, Chen Keli, Research on DNA barcoding identification of Qianhu and its counterfeit products, Chinese Materia Medica, 2013, 36(11 ): 1762), but this method takes a long time and uses large instruments, which is not conducive to rapid and on-site detection

Method used

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  • Primer pair used for identifying peucedanum praeruptorum and application thereof
  • Primer pair used for identifying peucedanum praeruptorum and application thereof
  • Primer pair used for identifying peucedanum praeruptorum and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Preparation and use method of kit for identifying Peucedanum praeruptorum

[0039] 1. Design and Synthesis of Primer Pairs Used to Identify Peucedanum

[0040] Find the ITS sequences of Peucedanum and its confounders from GenBank, see Table 1 below for details.

[0041] Table 1 ITS sequence of Peucedanum and its confounding products

[0042]

[0043]

[0044] Bayesian inference (BI) and maximum parsimony (MP) were used for phylogenetic analysis to construct BI and MP phylogenetic trees. The BI tree was constructed with MrBayes version 3.1.2 software, and the MP tree was constructed with PAUP*version 4.0beta 10 software. When constructing the BI tree, use MrModeltest version 2.3 software to select the optimal model of the data according to the AIC (Akaike Information Criterion) test standard, and the optimal model selected is (GTR+I+G). The Markov Chains Monte Carlo method (Markov Chains Monte Carlo, MCMC) is set to four chains and runs 500,000 generations. In orde...

Embodiment 2

[0060] Example 2. Using the kit prepared in Example 1 to identify the specificity analysis of Peucedanum praeruptorum

[0061] Samples to be tested: Qianhu (collected from Ningguo, Anhui), Zihua Qianhu (collected from Jinzhai, Anhui). All comply with the relevant regulations of each medicinal material in a main text of the Chinese Pharmacopoeia (2010 edition). Through identification, the actual product of each medicinal material is consistent with the name, and the quality meets the standard.

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

[0063] Take about 50 mg of dry samples (of course, 100 mg of fresh samples can also be used for genomic DNA extraction), and extract total DNA by CTAB method. details as follows:

[0064] Place the mildew-free dry medicinal materials in a pulverizer to grind and smash, and pass through a 40-mesh sieve. Transfer the powder to a 2.0mL microcentrifuge tube, add 900μL of sterilized CTAB extract (recipe: 2% (2g / 100ml) CTAB, 100mmol / L Tr...

Embodiment 3

[0075] Example 3. Sensitivity analysis for the identification of Peucedanum praeruptorum using the kit prepared in Example 1

[0076] Sample to be tested: Qianhu (collected from Ningguo, Anhui). All comply with the relevant regulations of each medicinal material in a main text of the Chinese Pharmacopoeia (2010 edition). Through identification, the actual products of the medicinal materials are consistent with the name, and the quality meets the standards.

[0077] 1. Extract genomic DNA from the sample to be tested

[0078] Take about 50 mg of dry samples (of course, 100 mg of fresh samples can also be used for genomic DNA extraction), and extract total DNA by CTAB method. For specific operations, refer to step one in embodiment 2.

[0079] 2. PCR amplification

[0080] The genomic DNA extracted from Qianhu in step 1 was diluted by multiples, and the genomic DNA concentrations were 42ng / μl, 21ng / μl, 11ng / μl, 5ng / μl, 2.5ng / μl, 1.25ng / μl and 0.625ng / μl serial dilutions, using the se...

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Abstract

The invention discloses a primer pair used for identifying peucedanum praeruptorum and application thereof. The primer pair used for identifying peucedanum praeruptorum or assisting the identification of peucedanum praeruptorum is specifically a primer pair formed by two single-chain DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table (shown in the description). An experiment result proves that through the adoption of the primer pair provided by the invention, the quick and accurate identification between the peucedanum praeruptorum and counterfeit species like peucedanum decursivum is realized through a quick PCR method, and technical support is provided for the site utilization of medicinal material molecule identification.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology, relates to the field of identification of traditional Chinese medicines and traditional Chinese medicinal materials, and particularly relates to a primer pair for identifying Qianhu and its application. Background technique [0002] Peucedanum is the dry root of Peucedanum praeruptorum Dunn. Due to the different medication habits in various regions in history, the plant source of Peucedanum praeruptorum is more complicated and there are many fake products. The main fake products on the market are Peucedanum praeruptorum, Peucedanum crassipes, Peucedanum chinense, Ligusticum chinense, etc. Leaf anise, Shi Fangfeng, short film Gao Ben, etc. [0003] The morphological characteristics of Qianhu medicinal materials are similar to those of the same family or the same genus, and it is difficult to identify the appearance. The traditional identification methods have certain limitations. Therefore, i...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 黄璐琦袁媛赵群
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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