CN114182002A - Method for detecting pseudo-ginseng in Chinese patent medicine and application - Google Patents
Method for detecting pseudo-ginseng in Chinese patent medicine and application Download PDFInfo
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- CN114182002A CN114182002A CN202111383408.8A CN202111383408A CN114182002A CN 114182002 A CN114182002 A CN 114182002A CN 202111383408 A CN202111383408 A CN 202111383408A CN 114182002 A CN114182002 A CN 114182002A
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Abstract
The invention provides a method for detecting pseudo-ginseng in a Chinese patent medicine. Specific primers and TaqMan probes are designed based on ITS2 sequences of pseudo-ginseng, a fluorescent quantitative PCR reaction system is constructed, pseudo-ginseng and common mixed counterfeit products of pseudo-ginseng can be distinguished, and the fluorescent quantitative PCR reaction system is used for detecting pseudo-ginseng-containing Chinese patent medicines in the market. The specific primer and the TaqMan probe are sequences shown in SEQ ID No. 1-3. The method has strong specificity, high sensitivity and good repeatability, and can be used as a powerful means for quality control and market supervision of Chinese patent medicines to ensure safety and effectiveness of Chinese medicines.
Description
Technical Field
The invention belongs to the technical field of identification of Chinese medicinal material source species, and particularly relates to a method for specifically detecting pseudo-ginseng in Chinese patent medicines based on TaqMan probe fluorescent quantitative PCR reaction.
Background
The Chinese pharmacopoeia contains dried root and rhizome of Panax notoginseng (Burk.) F.H.Chen of Araliaceae. Pseudo-ginseng is one of the traditional Chinese medicinal materials, and has the effects of removing blood stasis, stopping bleeding, reducing swelling and relieving pain. Clinical research shows that the traditional Chinese medicine composition has obvious effect on preventing and treating cardiovascular and cerebrovascular diseases. The Chinese patent medicine containing pseudo-ginseng occupies a certain share of the market of Chinese patent medicines. According to statistics, 513 Chinese patent medicine preparations taking pseudo-ginseng as a raw material exist in China at present, and the batch of the medicines containing the pseudo-ginseng raw material is as many as 3600. The Chinese patent medicine is directly applied to clinic, and the safety and the effectiveness of the Chinese patent medicine influence the clinical medication safety, so the quality control and the supervision of the Chinese patent medicine are very important.
The Chinese patent medicine has complex prescription and contains various traditional Chinese medicinal materials. The foundation is the source for ensuring the quality, and the identification items of the existing Chinese patent medicine quality control method comprise microscopic identification and physicochemical identification. The operation is complicated and needs to be supported by professional knowledge or large-scale instruments and equipment. Modern molecular identification technologies such as DNA barcodes, molecular identity cards and the like all comprise a DNA sequencing step, the time consumption is long, and the DNA of Chinese patent medicine is degraded to different degrees through preparation process treatment, so that the target fragment is often difficult to successfully amplify. The TaqMan probe fluorescent quantitative PCR method realizes species identification by combining a specific primer, a probe and a target fragment to generate a fluorescent signal. Compared with other molecular techniques, the probe has strong specificity, can generate a fluorescent signal only when the probe recognizes a complementary sequence, and can be used for distinguishing species with highly similar sequences; the target fragment is short, and the DNA degradation is still applicable; the sensitivity is high, and the method is suitable for a mixed system; does not need a DNA sequencing step and is suitable for occasions needing to obtain results quickly, such as market sampling inspection and the like. Therefore, the detection method of the Chinese patent medicine containing the pseudo-ginseng can be established based on the detection method, so as to be used for quality control and market supervision of the Chinese patent medicine containing the pseudo-ginseng and ensure the safety and effectiveness of the Chinese patent medicine.
Disclosure of Invention
The invention aims to provide a fluorescent quantitative PCR primer and a TaqMan probe for specifically identifying pseudo-ginseng.
The second purpose of the invention is to provide a fluorescent quantitative PCR method for detecting pseudo-ginseng in Chinese patent medicine.
The third purpose of the invention is to provide the application of the specific primer and the TaqMan probe in detecting the pseudo-ginseng in the Chinese patent medicine.
The invention provides a method for detecting pseudo-ginseng in a Chinese patent medicine, which is characterized by comprising the following steps: the method for detecting the pseudo-ginseng in the Chinese patent medicine is carried out based on TaqMan probe fluorescent quantitative PCR reaction.
The invention provides a method for detecting pseudo-ginseng in a Chinese patent medicine, which is characterized by comprising the following steps: the TaqMan probe-based fluorescent quantitative PCR reaction comprises a specific primer and a TaqMan probe, the specific primer and the TaqMan probe are used for identifying pseudo-ginseng and pseudo-mixed products thereof, detected samples are pseudo-ginseng medicinal materials, Chinese patent medicines containing pseudo-ginseng, related health products and formulated preparations, and nucleotide sequences of the specific primer and the TaqMan probe are shown in a table 1:
TABLE 1
The invention provides a method for detecting pseudo-ginseng in a Chinese patent medicine, which is characterized by comprising the following steps:
1) extracting genome DNA of a sample to be detected;
2) performing fluorescent quantitative PCR amplification by using the fluorescent quantitative PCR specific primer and the TaqMan probe of claim 1 by using a genome DNA of a sample to be detected as a template;
3) and judging whether the sample contains the pseudo-ginseng or not according to the amplification curve.
Preferably, the extraction of the genomic DNA of the sample to be tested in the step 1) comprises the following steps: according to the Chinese patent medicine preparation, different pretreatment modes are adopted: scraping the coating of the tablet containing the coating by using a sterilizing blade; ② the capsule shell should be removed; ③ the suppository should be melted by metal bath or water bath. After pretreatment, the sample is rinsed for 3 times by using a nucleic acid separation solution and then extracted by using a plant genome DNA extraction kit; the fluorescent quantitative PCR reaction condition parameters in the step 2) are as follows: at 95 ℃ for 30 s; (95 ℃, 5s, 60 ℃, 30s) for 40 cycles, and collecting fluorescence signals; the total volume of the amplification reaction is 20 muL, wherein the volume of the Probe qPCR Mix (2x) is 10 muL, the volume of each of the upstream primer and the downstream primer (10 muM) is 0.4 muL, the volume of each of the Probe (10 muM) and the template DNA is 2 muL, and the volume is made up to 20 muL by sterilized distilled water; the judgment condition in the step 3) is that the Ct value is less than 30.
The present invention will be further explained with reference to the drawings to fully explain the objects, technical features and technical effects of the present invention.
Drawings
FIG. 1 shows the specific detection results of fluorescent quantitative PCR primers and TaqMan probes for Notoginseng radix.
FIG. 2 shows the result of the sensitivity detection of fluorescent quantitative PCR reaction of Notoginseng radix.
FIG. 3 shows the result of fluorescence quantitative PCR detection of Notoginseng radix in Chinese patent medicine.
Detailed Description
The present invention is further described with reference to the following specific examples, which are not intended to be limiting, but rather are intended to be exemplary of the invention in which the prior art is known and in which the invention is not limited only to the extent that certain equivalents are known in the art, and therefore all are intended to be encompassed within the scope of the invention.
Example 1
1) DNA extraction
Cleaning Notoginseng radix, Ginseng radix, radix Panacis Quinquefolii, and radix Curcumae sample with 75% alcohol cotton ball, air drying, weighing 40-100mg, grinding with MM400 tissue grinder (Retsch, Germany) for 2min (30 times/s), and extracting genome DNA according to the instruction of plant genome DNA extraction kit (DP305, Tiangen Biochemical technology (Beijing) Co., Ltd.). DNA concentration was determined using Nanodrop 2000.
2) Fluorescent quantitative PCR reaction
The fluorescent quantitative PCR reaction system is 20 μ L, wherein the Probe qPCR Mix (2X) is 10 μ L, the upstream and downstream primers (10 μ M) are 0.4 μ L each, the Probe (10 μ M) is 0.8 μ L, the template DNA is 2 μ L, and the volume is made up to 20 μ L with sterilized distilled water.
Carrying out fluorescent quantitative PCR reaction on the sample by adopting a BioRad CFX96 fluorescent quantitative PCR instrument, wherein the reaction conditions are as follows: at 95 ℃ for 30 s; (95 ℃, 5s, 60 ℃, 30s), 40 cycles, and collecting the fluorescence signal.
3) Specificity of fluorescent quantitative PCR reaction
The fluorescent quantitative PCR detection result shows that the pseudo-ginseng amplification curve is S-shaped, and is a typical positive reaction; no specific amplification curve is seen in the 3 mixed counterfeit products of ginseng, American ginseng and turmeric root-tuber, and the reaction is negative; the blank control sterilized distilled water has no specific amplification curve and is a negative reaction. In conclusion, the identification method has strong specificity and can effectively identify the pseudo-ginseng and common adulterants thereof.
Example 2
1) DNA extraction
Basically, the same as example 1, except that only the genomic DNA of Panax notoginseng was extracted. The extracted genomic DNA was diluted with sterilized distilled water to 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 0.1 ng/. mu.L, 0.01 ng/. mu.L, 0.001 ng/. mu.L, 0.0001 ng/. mu.L, 0.00001 ng/. mu.L.
2) Fluorescent quantitative PCR reaction
The same as in example 1.
3) Fluorescent quantitative PCR reaction sensitivity
The fluorescent quantitative PCR detection result shows that when the concentration of the template DNA is 100-0.001 ng/. mu.L, good specific fluorescent amplification curves can be generated, the Ct value is less than 30, and when the concentration is 0.0001-0.00001 ng/. mu.L, the amplification curves do not present a typical 'S' shape, and the Ct value is more than 30, so that the sensitivity of the identification method is high, and the minimum detection limit is 0.001 ng/. mu.L.
Example 3
1) DNA extraction
Basically the same as example 1, except that 75 parts of Chinese patent medicine containing notoginseng are collected and extracted for genome DNA. The DNA is pretreated according to the Chinese patent medicine preparation before extraction: firstly, coating of the coated tablet needs to be scraped by a sterilization blade; ② the capsule shell should be removed; ③ the suppository should be melted by metal bath or water bath. After sampling, the nucleic acid separation solution should be rinsed 3 times and then the operation should be performed according to the instructions of the plant genome DNA extraction kit. The formula of the nucleic acid separating solution is as follows: 20mM EDTA (pH 8.0), 100mM Tris-HCl (pH 8.0), 700mM NaCl, 2% PVP-40, 0.4% beta-mercaptoethanol.
TABLE 2 sample information Table
2) Fluorescent quantitative PCR reaction
As in example 1.
3) Fluorescent quantitative PCR detection result
The result of the fluorescent quantitative PCR detection shows that all sample amplification curves are S-shaped, the Ct value is less than 30, and the sample amplification curves are typical positive reactions.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications can be made without departing from the technical principle of the present invention, and these modifications should also be regarded as the protection scope of the present invention.
Claims (4)
1. A method for detecting pseudo-ginseng in Chinese patent medicine is characterized by comprising the following steps: the method for detecting the pseudo-ginseng in the Chinese patent medicine is carried out based on TaqMan probe fluorescent quantitative PCR reaction.
2. The method for detecting pseudo-ginseng in Chinese patent medicine according to claim 1, wherein the method comprises the following steps: the TaqMan probe-based fluorescent quantitative PCR reaction comprises a specific primer and a TaqMan probe, the specific primer and the TaqMan probe are used for identifying pseudo-ginseng and pseudo-mixed products thereof, detected samples are pseudo-ginseng medicinal materials, Chinese patent medicines containing pseudo-ginseng, related health products and formulated preparations, and nucleotide sequences of the specific primer and the TaqMan probe are shown in a table 1:
TABLE 1
3. The method for detecting pseudo-ginseng in Chinese patent medicine according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
1) extracting genome DNA of a sample to be detected;
2) performing fluorescent quantitative PCR amplification by using the fluorescent quantitative PCR specific primer and the TaqMan probe of claim 1 by using a genome DNA of a sample to be detected as a template;
3) and judging whether the sample contains the pseudo-ginseng or not according to the amplification curve.
4. The method for detecting pseudo-ginseng in Chinese patent medicine according to claim 3, wherein the method comprises the following steps: the extraction of the genome DNA of the sample to be detected in the step 1) comprises the following steps: according to the Chinese patent medicine preparation, different pretreatment modes are adopted: scraping the coating of the tablet containing the coating by using a sterilizing blade; ② the capsule shell should be removed; ③ the suppository should be melted by metal bath or water bath. After pretreatment, the sample is rinsed for 3 times by using a nucleic acid separation solution and then extracted by using a plant genome DNA extraction kit; the fluorescent quantitative PCR reaction condition parameters in the step 2) are as follows: at 95 ℃ for 30 s; (95 ℃, 5s, 60 ℃, 30s), 40 cycles, and collecting the fluorescence signal.
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| CN202111383408.8A CN114182002A (en) | 2021-11-22 | 2021-11-22 | Method for detecting pseudo-ginseng in Chinese patent medicine and application |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6803215B1 (en) * | 2000-11-03 | 2004-10-12 | The Chinese University Of Hong Kong | Sequence characterized amplified region (SCAR) test for the authentication of traditional Chinese medicinal materials |
| CN102888456A (en) * | 2012-09-21 | 2013-01-23 | 中国医学科学院药用植物研究所 | Method for quickly identifying pseudo-ginseng |
| CN104830969A (en) * | 2015-03-17 | 2015-08-12 | 中国医学科学院药用植物研究所 | Panax notoginseng molecule ID and identification method |
| CN106701955A (en) * | 2017-01-09 | 2017-05-24 | 中国中医科学院中药研究所 | Primer combination for simultaneously identifying Chinese herb ginseng, American ginseng and radix notoginseng and application of primer combination |
| CN112322766A (en) * | 2020-10-23 | 2021-02-05 | 中国检验检疫科学研究院 | Accurate pseudo-ginseng powder quantitative method based on micro-drop digital PCR technology |
-
2021
- 2021-11-22 CN CN202111383408.8A patent/CN114182002A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6803215B1 (en) * | 2000-11-03 | 2004-10-12 | The Chinese University Of Hong Kong | Sequence characterized amplified region (SCAR) test for the authentication of traditional Chinese medicinal materials |
| CN102888456A (en) * | 2012-09-21 | 2013-01-23 | 中国医学科学院药用植物研究所 | Method for quickly identifying pseudo-ginseng |
| CN104830969A (en) * | 2015-03-17 | 2015-08-12 | 中国医学科学院药用植物研究所 | Panax notoginseng molecule ID and identification method |
| CN106701955A (en) * | 2017-01-09 | 2017-05-24 | 中国中医科学院中药研究所 | Primer combination for simultaneously identifying Chinese herb ginseng, American ginseng and radix notoginseng and application of primer combination |
| CN112322766A (en) * | 2020-10-23 | 2021-02-05 | 中国检验检疫科学研究院 | Accurate pseudo-ginseng powder quantitative method based on micro-drop digital PCR technology |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: KP218740.1: "Panax notoginseng isolate 18 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence", 《GENBANK》 * |
| QIAN LOU ET AL.: "TaqMan Probe-Based Quantitative Real-Time PCR to Detect Panax notoginseng in Traditional Chinese Patent Medicines", 《FRONT PHARMACOL》, pages 828948 * |
| 王巍 等: "市售三七粉中三七的DNA检测方法研究", 《时珍国医国药》, pages 1400 - 1401 * |
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