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A kind of primer pair and application thereof for identifying long-distance dendrobium

A technology for primer pair and long identification is applied in the field of primer pair identification of long-distance dendrobium to achieve the effect of accurate identification

Active Publication Date: 2017-12-22
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

[0004] Rapid, accurate, and high-throughput identification is the core requirement for molecular identification of modern Chinese medicines. At present, specific PCR methods have been successfully used in the identification of some Dendrobium plants, such as Dendrobium spp. Using plant authenticity identification (Luo Yuming, Ding Xiaoyu, Xu Luoshan, et al. Site-specific PCR identification of Dendrobium slender leaves, Journal of Huaiyin Normal University, 2002, 1(1):82-85; Xu Hong, Wang Zhengtao, Ding Xiaoyu , et al, Study on site-specific PCR identification of Dendrobium nobile, Chinese Herbal Medicine, 2005, 36(10):1540-1544; Liu Shiquan, Li Xiaojun, Yu Qingbo, et al. Site-specific PCR identification of Dendrobium nobile and similar species, Chinese Herbal Medicine, 2006, 37(1):111-114; Ding Xiaoyu, Xu Luoshan, Chang Jun, et al. Site-specific PCR identification of Dendrobium dendrobii, Journal of Nanjing Normal University, 2002, 25(4):71-75), and Dendrobium longifolia has no related reports

Method used

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  • A kind of primer pair and application thereof for identifying long-distance dendrobium
  • A kind of primer pair and application thereof for identifying long-distance dendrobium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, the preparation and the use method of the kit for identifying the dendrobium long-distance

[0050] 1. Design and synthesis of primer pairs for identification of long-distance Dendrobium

[0051] 本实施例根据GenBank中长距石斛及其同属近缘种的psbA-trnH序列(GenBank登录号:KF177467,KF177469,KF177471,KF177472,EU672793,KF177480,KF177482,KF177483,JF693815,KF177486,KF177493,KF177494,KF177500 ,KF177501,FJ216470,KF177506,KF177507,KF177509,KF177511,KF177520,KF177521,KF177522,KF177526,KF177535,KF177540,KF177538,KF177554,EU887927),进行同源比对,找出长距石斛的稳定变异位点,根据 A pair of specific primers F1 and F2 are designed for the variable region, and the sequence is as follows:

[0052] F1: 5'-GGCTAACACTTTTGCTTCTCTATAT-3' (SEQ ID NO: 1);

[0053] F2: 5'-GACTTGGTCTCGGGCATCTA-3' (SEQ ID NO: 2).

[0054] 2. Assembly of kits for identification of Dendrobium longifolia

[0055] After the primers F1 and F2 designed and synthesized in step 1 are packaged separately, they are packaged in the same kit with separately ...

Embodiment 2

[0068] Embodiment 2, using the kit prepared in Example 1 to identify Dendrobium long-distance

[0069] Samples to be tested: 16 species of Dendrobium shown in Table 1.

[0070] 1. Extract genomic DNA from the sample to be tested

[0071]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 chloroform-isoamyl alcohol (volume ratio 24:1), shake and mix well, and centrif...

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Abstract

The invention discloses a primer pair for identifying dendrobium longicornu and an application of the primer pair. The primer pair for identifying or helping to identify the dendrobium longicornu is specifically composed of two single-chain DNA molecules as shown in a sequence 1 and a sequence 2 in the sequence table. Experiments prove that the primers are adopted to realize quick and accurate identification of the dendrobium longicornu and the congeneric sibling species thereof by use of a fast PCR method and a fluorescent staining method; and a fluorescent dye method is introduced to detect the true and false identification results, and technical support is provided for the field application of molecular identification of medicinal materials.

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 medicinal materials, in particular to a pair of primers for identifying long-distance Dendrobium dendrobium and its application. Background technique [0002] Dendrobium is a commonly used famous and precious traditional Chinese medicine, which has been recorded in many herbal medicines in the past dynasties, and is listed as the top grade in "Shen Nong's Materia Medica". Dendrobium is sweet in nature, slightly cold, and returns to the stomach and kidney meridian. It has the effects of nourishing the stomach, nourishing yin and clearing away heat. , hyperactivity of fire due to deficiency of yin, bone steaming and consumptive fever, dark eyesight, weakness of muscles and bones (Yu Meijuan, Meng Qinghua, Huang Deyin, etc. Research on the active ingredients and pharmacological effects of Dendrobium plants, Chinese...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2563/107
Inventor 黄璐琦袁媛崔占虎蒋超
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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