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A kit and ginseng technology, applied in the field of molecular biology, can solve problems such as unfavorable application and promotion, cumbersome fluorescent staining methods, and long testing time
Inactive Publication Date: 2015-03-25
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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[0004] To sum up, different researchers have used RAPD method, AP-PCR method, PCR-SSCP method, multiplex PCR method, etc. to identify other medicinal materials in Panax ginseng, but these methods take a long time to test and require Electrophoresis detection can only be completed, so compared with the fluorescent staining method, it is cumbersome, which is not conducive to the application and promotion of on-site rapid identification
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Embodiment 1
[0032] Embodiment 1, the preparation and use method of the kit for identifying ginseng
[0033] 1. Design and synthesis of primer pairs for identification of ginseng
[0034] In this example, the general primer 18S rRNA (18SF: 5'-CAACCTGCTTGATCCTGCCAGT-3'18SR: 5'-CTGATCCTTTCTGCAGGTTCACCTA-3') was selected to amplify the samples of Panax ginseng, Panax notoginseng, Panax notoginseng, Pearl ginseng and Panax notoginseng. Amplified, sequenced, and then homologously compared the obtained sequences, and designed a pair of specific primers F1 and F2 according to the variable region, as a pair of specific primers for identifying ginseng. The sequence is as follows:
[0035]Primer F1: 5'-ATAACAATACCGGGCTGATTC-3' (SEQ ID NO: 1);
[0036] Primer F2: 5'-GCCAGTTAAGGACAGGAG-3' (SEQ ID NO: 2).
[0037] 2. Assembly of kits for identification of ginseng
[0038] After the primers F1 and F2 designed and synthesized in step 1 are packaged separately, they are packaged in the same kit with s...
Embodiment 2
[0051] Embodiment 2, using the kit prepared in embodiment 1 to identify ginseng
[0052] Samples to be tested: 6 samples of Chinese herbal medicines shown in Table 1, ginseng, American ginseng, Panax notoginseng, bamboo ginseng, pearl ginseng, and Panax notoginseng.
[0053] 1. Extract genomic DNA from the sample to be tested
[0054] 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, ad...
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Abstract
The invention discloses a ginseng identification method and a special kit. The method comprises the steps: a) extracting genomes DNA as templates from a to-be-tested sample and utilizing primers (sequence 1 and sequence 2) to perform PCR (Polymerase Chain Reaction) amplification; b) adding a fluorescent dye SYBR Green I to the amplified reaction system and determining whether the to-be-tested sample contains ginsengs according to the following methods; if green fluorescence is observed in the reaction system, the to-be-tested sample or the candidate product contains ginsengs; if no green fluorescence is observed in the reaction system, the to-be-tested sample or the candidate product does not contain ginsengs. The experimental results show that the rapid and accurate identification of ginsengs, American ginsengs, pseudo-ginsengs, panax japonicus, rhizoma panacis majoris and bipinnatifid ginseng rhizome can be realized by the method provided by the invention; the fluorescent dye method is introduced to detect true or false identification results and technical supports are provided to realize onsite application of identification of medicinal molecules.
Description
technical field [0001] The invention belongs to the field of molecular biology, and relates to a ginseng identification method and a special kit. Background technique [0002] The identification methods of Panax genus herbal medicinal materials mainly include morphological identification, physical and chemical identification, cell identification, etc. These traditional identification methods are easily affected by environmental conditions or limited by the plant itself, and there are many human interference factors, so they cannot directly reflect the genetics of the germplasm. characteristics, sometimes it is difficult to get accurate results (Zhou Yanqing. Application of DNA molecular marker technology in plant research. Beijing: Chemical Industry Press, 2005.). [0003] DNA molecular marker technology is a new detection technology that has emerged in the past two decades and has been continuously developed and improved. In recent years, DNA molecular marker technology ha...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2563/107
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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