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Specific primer, kit and identification method for identifying poria cocos

A kit and specific technology are applied in the field of identifying specific primers for Poria cocos, which can solve the problems of low accuracy and affect the PCR amplification efficiency, and achieve the effects of high accuracy, good specificity and easy mastery.

Pending Publication Date: 2022-05-06
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The counterfeit products of Poria cocos on the market are mostly processed from starch and plant powder. It is easier to distinguish whether there is starch in the decoction pieces by using physical and chemical methods.
The DNA barcoding technology relies on the gene molecular information of the sample itself, and can be used to identify whether the sample is Poria cocos, but the content of polysaccharides in Poria cocos is high, especially for common Chinese medicines containing Poria cocos such as Poria decoction pieces and Guizhi Fuling pills. Processing processes such as drying and drying will lead to serious degradation of the DNA of the sample, which not only affects the efficiency of PCR amplification, but also lowers the accuracy of amplification and sequencing

Method used

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  • Specific primer, kit and identification method for identifying poria cocos
  • Specific primer, kit and identification method for identifying poria cocos
  • Specific primer, kit and identification method for identifying poria cocos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] 1. Experimental materials

[0052] In this case, a total of 13 Poria cocos samples were collected, of which 12 (No. 1-12) were commercially available Poria cocos decoction pieces, and 1 (No. 13) was fresh Poria cocos (as a positive control). The specific information is shown in Table 1.

[0053] Table 1 Poria cocos sample information table

[0054]

[0055] 2. Experimental equipment

[0056] MM400 ball mill (Retsch, Germany), desktop high-speed centrifuge (SIGMA1-14, Germany), PCR instrument (Biometra Tone, Germany), electrophoresis instrument (DYY-8C, Beijing Liuyi Instrument Factory), gel imager (BIO-RAD, USA).

[0057] 3. DNA extraction

[0058] The above 13 samples were subjected to DNA extraction, and the extraction method was as follows: take 100 mg of dried Poria cocos, pulverize it with a MM400 ball mill, add 1,000 μL of cleavage solution, mix well, centrifuge at 7,500 r / min for 3 min, remove the supernatant, and repeat 3-5 times. Repeat until the supernata...

Embodiment 2

[0076] The present embodiment provides a kind of method for identification Poria cocos composition in fresh Poria cocos, comprises the steps:

[0077] (1) DNA extraction

[0078] Take fresh Poria cocos as the sample to be tested, and extract it. The extraction method is as follows: take 40 mg of the sample to be tested, pulverize it with a MM400 ball mill, add 1,000 μL of cleavage solution, mix well, centrifuge at 7,500 r / min for 3 minutes, remove the supernatant, Repeat 3-5 times until the supernatant is not viscous. The precipitate was taken, and the rest of the steps were extracted according to the standard operation method of the DNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to obtain the DNA template. The formula of nuclear isolation liquid is: Tris-HCl (pH8.0) final concentration 100mM, EDTA (pH8.0) final concentration 20mM, NaCl final concentration 0.7M, 2%PVP-40 (W / V), above each material formulating Sterilize after forming a solution, ...

Embodiment 3

[0088] 1. Experimental materials

[0089] In this case, a total of 3 samples of Guizhi Fuling pills were collected, and the specific information is shown in Table 2.

[0090] Table 2 Sample information of Guizhi Fuling Pills

[0091]

[0092] 2. Experimental steps

[0093] (1) DNA extraction

[0094] Take Guizhi Fuling Pills as the sample to be tested, and extract it. The extraction method is as follows: Take 100 mg of the sample to be tested, pulverize it with a MM400 ball mill, add 1,200 μL of cleavage solution, mix well, centrifuge at 7,500 r / min for 3 minutes, and remove the upper layer solution , repeat 3-5 times until the upper solution is colorless. Take the precipitate, and extract the precipitate according to the standard operation method of the DNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to obtain the DNA template. The formula of nuclear isolation liquid is: Tris-HCl (pH8.0) final concentration 50mM, EDTA (pH8.0) final concentr...

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Abstract

The invention provides specific primers, a kit and an identification method for identifying poria cocos, and the primers are as follows: a forward primer is 5 '-CTCCTCCAAACGCATTAGC-3'; and the reverse primer is 5 '-AATCTGTTTGTCCATCGACC-3'. A plurality of pairs of primers are designed on the basis of the ITS gene of the poria cocos, the optimal primer with species specificity is screened out through a large number of primer screening studies, and effective amplification of a target gene can be achieved by using the primer pairs, so that the poria cocos can be rapidly and accurately identified.

Description

technical field [0001] The invention belongs to the technical field of identification of traditional Chinese medicinal materials, and in particular relates to a specific primer, a kit and an identification method for identification of poria cocos. Background technique [0002] Poria cocos is the dried sclerotia of Polyporaceae fungus Poria cocos (Schw.) Wolf. Sweet, light, neutral in nature, it has the effects of diuresis and dampness, invigorating the spleen and stomach, calming the mind and calming the nerves. Modern studies have shown that Poria cocos mainly contains two compounds, triterpenes and polysaccharides, which have pharmacological effects such as diuresis, anti-inflammatory and liver protection. As one of the eight treasures of traditional Chinese medicine, Poria cocos is also widely used in food. Accurate identification of Poria cocos not only ensures the safety of clinical medication, but also has great significance for regulating the market of Chinese herba...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11C12Q1/686
CPCC12Q1/6895C12Q1/686C12Q2527/125
Inventor 姚辉王亮王清王勇武立伟韩正洲吴正军马鹏岗宋经元聂丽萍
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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