CN102827940A - PCR (polymerase chain reaction) detection method for ginseng DNA (deoxyribonucleic acid) in Chinese patent medicine - Google Patents
PCR (polymerase chain reaction) detection method for ginseng DNA (deoxyribonucleic acid) in Chinese patent medicine Download PDFInfo
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Abstract
The invention relates to the field of analysis detection and discloses a PCR (polymerase chain reaction) detection method for ginseng DNA (deoxyribonucleic acid) in Chinese patent medicine for identifying the authenticity of ginseng in the Chinese patent medicine. The method concretely comprises a set of detection operation programs including ginseng DNA extraction in the Chinese patent medicine, extracted nucleic acid amplification, amplification product sepharose gel detection, result judgment and the like. On the basis of the ordinary PCR, the nest type PCR reaction method is built, the ginseng in the Chinese patent medicine can be fast, accurately and sensitively detected, and the single, fast and effective method is provided for the quality control of the Chinese patent medicine.
Description
Technical field
The invention belongs to the authentication method of Chinese patent medicine true or false, specifically utilize round pcr to detect a kind of method of the DNA of genseng in the Chinese patent medicine.
Background technology
The quality control of traditional Chinese patent medicine only limits to the assay to discriminating on the property of drug and compound.Chinese medicinal materials is processed Chinese patent medicine, and the proterties of itself had been difficult to distinguish when crude drug was processed Chinese patent medicine through several formulations technology; And the complicated component of Chinese medicine own, Herba indigoferae Pseudotinctoriae is with regard to tens even hundreds of compounds, a Chinese patent medicine of being made up of plurality of Chinese; Wherein chemical ingredients is just more, only selects the evaluation of several kinds of limited chemical ingredientss to control the quality of Chinese patent medicine at present, just for the Chinese patent medicine fake producer very big space is provided; For certain expensive Chinese medicinal materials, for example genseng does not add the ginseng crude drug in the preparation process; Only add the compound of being classified as test item, perhaps pass a fake product off as a genuine one with other medicinal materials that contain same compound, present method is through the amplification of PCR; To still detecting the wherein DNA of genseng through the Chinese patent medicine of beating after powder, extraction, the filtration; Overcome the deficiency of prior art, can judge whether use genseng in the Chinese patent medicine easy, quickly and accurately, ensured drug quality.
Summary of the invention
The purpose of this invention is to provide the method that a kind of PCR detects genseng DNA in the Chinese patent medicine.
Aforesaid method of the present invention is to utilize that the specific DNA of genseng detects the genseng DNA in the Chinese patent medicine in polymerase chain reaction (PCR) the amplification Chinese patent medicine.Wherein the Chinese patent medicine that detects is to contain the Chinese patent medicine that the Latin formal name used at school is Panax ginseng C.A.Mey., its another name or its processed product.
Aforesaid method of the present invention comprises the following steps:
1) extraction of genseng DNA in the Chinese patent medicine;
2) PCR reaction;
3) pcr amplification product analysis.
In the aforesaid method, the formulation of being used as medicine with medicinal powder for the Chinese patent medicine medicinal material in the step 1) adopts improved method of CTAB to extract DNA; The formulation of being used as medicine with extracting solution for the Chinese patent medicine medicinal material adopts the method for ethyl alcohol recrystallization to extract.
The formulation that wherein said medicinal powder is used as medicine comprises tablet, capsule and pill; The formulation that described extracting solution is used as medicine comprises oral liquid and injection liquid.
In the aforesaid method, step 2) adopt regular-PCR for the higher sample of dna content in, the sample lower for dna content adopts nest-type PRC.
The forward primer of wherein said common PCR reaction and reverse primer are to be the Auele Specific Primer of stencil design with genseng ITS2 sequence area, and its purpose band can be at 80-224bp; Described nest-type PRC carries out first round PCR and second successively to total genomic dna and takes turns twice amplification of PCR, and the dna fragmentation of first round pcr amplification product can be at 700-1500bp, and second to take turns PCR identical with regular-PCR.
Particularly, the method for genseng DNA comprises the following steps: in a kind of PCR detection Chinese patent medicine provided by the invention
1. the extraction of genseng DNA in the Chinese patent medicine:
Can select suitable process for extracting according to different dosage forms.
The medicine that medicinal powder is used as medicine comprises that formulations such as tablet, capsule, pill adopt improved method of CTAB to extract.1. medicinal material grinds to form fine powder in right amount and gets powder a small amount of (1, the minimum scale 0.1mL place of 5mL EP pipe) in 1.5mL EP pipe in mortar, adds the CTAB extraction damping fluid 700 μ l of 65 ℃ of preheatings, the mixing that fully vibrates, and 65 ℃ of temperature are bathed 2h; 2. sample adds chloroform-primary isoamyl alcohol (24 after being cooled to room temperature; 1) 600 μ l put upside down mixing 5 minutes; 3. 7500r.min
-1Centrifugal 10 minutes, shift in the new EP pipe of supernatant to; 4. the Virahol that adds equal-volume-20 ℃ precooling, mixing was placed 30 minutes for 20 ℃; 5. 10,000r.min
-1Centrifugal 15 minutes, abandon supernatant; 6. deposition is respectively washed once with 70% ethanol, absolute ethyl alcohol, and 10,000r.min
-1Centrifugal 3 minutes, abandon supernatant, room temperature volatilizes ethanol, and aseptic high purity water dissolving DNA is subsequent use.Said CTAB extracts damping fluid and contains: 2%CTAB (cetyl trimethylammonium bromide), 100mM Tris (pH 8.0), 20mMEDTA (pH 8.0), 2.5M NaCl.
Comprise oral liquid, injection liquid etc. that extracting solution is used as medicine adopt the method for ethyl alcohol recrystallization to extract.1. the 10mL soup adds 1mL 3M sodium-acetate (pH=5.2), the 30mL absolute ethyl alcohol, and 30 μ l glycogens (glycogen) are placed 1.5h for-20 ℃.2. 14,000r.min
-1Centrifugal 10 minutes, abandon supernatant.3. 70% washing with alcohol, 16,000r.min
-1Centrifugal 5 minutes, abandon supernatant.4. volatilize ethanol, aseptic high purity water dissolving DNA, subsequent use.
2. common PCR reaction system and condition:
10 * Taq buffer, 5 μ l; 5 units of Taq enzyme;
Forward primer 1 μ M; Reverse primer 1 μ M;
DNTPs 0.2mM; DNA extraction liquid 1 μ l.
95 ℃ were unwind 3 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of renaturation 30 seconds, 72 ℃ were extended 30 seconds, and circulated 30 times; 72 ℃ were extended 5 minutes, and sample must increase.
3. nest-type PRC reaction system and condition:
First round PCR:
10 * Taq buffer, 5 μ l; 5 units of Taq enzyme;
Forward primer 0.25 μ M; Reverse primer 0.25 μ M;
DNTPs 0.2mM; DNA extraction liquid 5 μ l.
95 ℃ were unwind 3 minutes; 94 ℃ of sex change 30 seconds, 60 ℃ of renaturation 30 seconds, 72 ℃ were extended 30 seconds, and circulated 25 times; 72 ℃ were extended 5 minutes, and sample must increase.
Second takes turns PCR operates with regular-PCR.
4.PCR amplified production analysis:
Get the sample after 5 μ l increase; Mix with 6 times of DNA sample-loading buffers of 1 μ l; On the agarose gel that contains the Gelview nucleic acid dye, carry out electrophoresis and colour developing, under the ultraviolet transmissive lamp, observe, sample well contain clauses and subclauses band for containing the positive of genseng or pollution.
The principle of the invention is: can extract the DNA of plant in the Chinese patent medicine through the method for CTAB method or ethyl alcohol recrystallization, contain polymerase chain reaction reagent in the PCR reaction reagent pipe, can carry out special amplification to the DNA of genseng in the reagent pipe; Owing to primer of the present invention is that genseng institute is peculiar; Therefore, if contain genseng in the sample, its DNA specific fragment is after the process amplification so; On the agarose gel that contains the Gelview nucleic acid dye, carry out electrophoresis and colour developing; UV-light detects just can detect the segmental purpose band of certain-length, if there is not genseng, and the segmental purpose band of the same length that then can not increase.
The present invention has the advantage that obviously is superior to prior art, and its major advantage comprises:
Novelty.Present method is in the discriminating of the method that first PCR the detected DNA true or false that is applied to Chinese patent medicine.
Rapidity.Adopt the inventive method to detect very fast, can learn detected result in 4-6 hour.
Susceptibility.Because the stability of DNA itself is not through still can pass through the amplification of PCR (polymerase chain reaction) exponential type again, and nest-type PRC being detected the DNA of denier as supplementary means by completely destroy after the processing of various preparation process.
Description of drawings
Fig. 1 is specific embodiment 1 detected result agarose gel figure; The wherein negative contrast of A, B is a tablet, the positive contrast of C, D is Marker.
Fig. 2 is specific embodiment 2 detected result agarose gel figure; The negative contrast of A, B is an injection, the positive contrast of C, D is Marker.
Embodiment
Below in conjunction with accompanying drawing, further specify the present invention through instance.One skilled in the art will understand that these instances only are used to explain the present invention, and be not used in restriction scope of the present invention.
The DNA of genseng in embodiment 1, the detection ginseng-astragalus sheet
According to following formulation genseng DNA extraction reagent
CTAB extracts damping fluid: 2%CTAB (cetyl trimethylammonium bromide), 100mMTris (pH 8.0), 20mM EDTA (pH 8.0), 2.5M NaCl.Virahol, chloroform, absolute ethyl alcohol, 70% ethanol, aqua sterilisa.
Detect according to follow procedure
1. the extraction of genseng control medicinal material DNA:
1. medicinal material grinds to form fine powder in right amount and gets powder a small amount of (the minimum scale 0.1mL place of 1.5mL EP pipe) in 1.5mL EP pipe in mortar, and the CTAB that adds 65 ℃ of preheatings extracts damping fluid 700 μ l, the mixing that fully vibrates, and 65 ℃ of temperature are bathed 2h; 2. sample adds chloroform one primary isoamyl alcohol (24 after being cooled to room temperature; 1) 600 μ l put upside down mixing 5 minutes; 3. 7500r.min
-1Centrifugal 10 minutes, shift in the new EP pipe of supernatant to; 4. the Virahol that adds equal-volume-20 ℃ precooling, mixing was placed 30 minutes for 20 ℃; 5. 10,000r.min
-1Centrifugal 15 minutes, abandon supernatant; 6. deposition is respectively washed once with 70% ethanol, absolute ethyl alcohol, and 10,000r.min
-1Centrifugal 3 minutes, abandon supernatant, room temperature volatilizes ethanol, and aseptic high water dissolution DNA is subsequent use.
2. the extraction of DNA of plants in the ginseng-astragalus sheet (with control medicinal material DNA extraction method)
3.PCR amplification
Forward primer: GGGGCGGATAATGGC
Reverse primer: ACGACATGAGAAGAGGGC
The PCR reaction system:
10 * Taq buffer, 5 μ l; 5 units of Taq enzyme;
Forward primer 1 μ M; Reverse primer 1 μ M;
DNTPs 0.2mM; DNA extraction liquid 1 μ l;
Ultrapure aqua sterilisa is supplied 50 μ l with system.
And control medicinal material DNA extraction liquid and ultrapure aqua sterilisa are set simultaneously respectively as positive control, negative control.
The PCR reaction conditions: 95 ℃ were unwind 3 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of renaturation 30 seconds, 72 ℃ were extended 30 seconds, and circulated 30 times; 72 ℃ were extended 5 minutes, and sample must increase.
4.PCR amplified production analysis
Send the order-checking of order-checking company with genseng control medicinal material PCR product, sequence is correct.
Get the sample after 5 μ l increase; Mix with 6 times of DNA sample-loading buffers of 1 μ l; On the agarose gel that contains the Gelview nucleic acid dye, carry out electrophoresis and colour developing; Under the ultraviolet transmissive lamp, observe, sample well contains a purpose band with the positive control same position to be explained in this medicine and has added the ginseng crude drug.
Detected result agarose gel figure is shown in accompanying drawing 1.
The DNA of genseng in embodiment 2, the detection SHENMAI ZHUSHEYE
The preparation of positive control and being provided with instance 1.
Detect according to follow procedure:
1. the extraction of genseng DNA in the SHENMAI ZHUSHEYE
1. the 10mL injection liquid adds 1mL 3M sodium-acetate (pH=5.2), the 30mL absolute ethyl alcohol, and 30 μ l glycogens (glycogen) are placed 1.5h for-20 ℃.2. 14,000r.min
-1Centrifugal 10 minutes, abandon supernatant.3. 70% washing with alcohol, 16,000r.min
-1Centrifugal 5 minutes, abandon supernatant.4. volatilize ethanol, aseptic high water dissolution DNA, subsequent use.
2. nest-type PRC
First round PCR:
Forward primer: CAC ACC GCC CGT CGC TCC TAC CGA
Reverse primer: ACT CGC CGT TAC TAG GGG AA
The PCR reaction system:
10 * Taq buffer, 5 μ l; 5 units of Taq enzyme;
Forward primer 0.25 μ M; Reverse primer 0.25 μ M;
DNTPs 0.2mM; DNA extraction liquid 1 μ l;
Ultrapure aqua sterilisa is supplied 50 μ l with system.
And control medicinal material DNA extraction liquid and ultrapure aqua sterilisa are set simultaneously respectively as positive control, negative control.The PCR reaction conditions: 95 ℃ were unwind 3 minutes; 94 ℃ of sex change 30 seconds, 60 ℃ of renaturation 30 seconds, 72 ℃ were extended 30 seconds, and circulated 25 times; 72 ℃ were extended 5 minutes, and sample must increase.
Second takes turns PCR
Forward primer: GGGGCGGATAATGGC
Reverse primer: ACGACATGAGAAGAGGGC
The PCR reaction system:
10 * Taq buffer, 5 μ l; 5 units of Taq enzyme;
Forward primer 1 μ M; Reverse primer 1 μ M;
DNTPs 0.2mM; First round PCR product 1 μ l;
Ultrapure aqua sterilisa is supplied 50 μ l with system.
And negative control and the positive control of getting first round moderate increase respectively as negative control, positive control simultaneously.
The PCR reaction conditions: 95 ℃ were unwind 3 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of renaturation 30 seconds, 72 ℃ were extended 30 seconds, and circulated 30 times; 72 ℃ were extended 5 minutes, and sample must increase.
3. nest-type PRC increases and product analysis
Get the sample after 5 μ l increase; Mix with 6 times of DNA sample-loading buffers of 1 μ l; On the agarose gel that contains the Gelview nucleic acid dye, carry out electrophoresis and colour developing; Under the ultraviolet transmissive lamp, observe, sample well contains a purpose band with the positive control same position to be explained in this medicine and has added the ginseng crude drug.
Detected result agarose gel figure is shown in accompanying drawing 2.
Claims (8)
1. the PCR detection method of genseng DNA in the Chinese patent medicine is characterized in that, said method is to utilize that the specific DNA of genseng detects the genseng DNA in the Chinese patent medicine in the pcr amplification Chinese patent medicine.
2. detection method according to claim 1 is characterized in that: the Chinese patent medicine that detects is to contain the Chinese patent medicine that the Latin formal name used at school is Panax ginseng C.A.Mey., its another name or its processed product.
3. detection method according to claim 1 and 2 is characterized in that said method comprises the following steps:
1) extraction of genseng DNA in the Chinese patent medicine;
2) PCR reaction;
3) pcr amplification product analysis.
4. method according to claim 3 is characterized in that, the formulation of being used as medicine with medicinal powder for the Chinese patent medicine medicinal material in the step 1) adopts improved method of CTAB to extract DNA; The formulation of being used as medicine with extracting solution for the Chinese patent medicine medicinal material adopts the method for ethyl alcohol recrystallization to extract.
5. method according to claim 4 is characterized in that the formulation that wherein said medicinal powder is used as medicine comprises tablet, capsule and pill; The formulation that described extracting solution is used as medicine comprises oral liquid and injection liquid.
6. method according to claim 3 is characterized in that, the sample higher for dna content adopts regular-PCR, and the sample lower for dna content adopts nest-type PRC.
7. method according to claim 6 is characterized in that, the forward primer of described common PCR reaction and reverse primer are to be the Auele Specific Primer of stencil design with genseng ITS2 sequence area, and its purpose band can be at 80-224bp.
8. method according to claim 6; It is characterized in that; Described nest-type PRC carries out first round PCR and second successively to total genomic dna and takes turns twice amplification of PCR, and the dna fragmentation of first round pcr amplification product can be at 700-1500bp, and second to take turns PCR identical with regular-PCR.
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| CN103131781A (en) * | 2013-02-28 | 2013-06-05 | 中国科学院成都生物研究所 | PCR detection method of DNA of biological counterfeit drug in Chinese patent drug |
| CN103173563A (en) * | 2013-04-15 | 2013-06-26 | 云南农业大学 | PCR-RFLP (Polymerase chain reaction-restriction fragment length polymorphism) method for quickly identifying panax notoginseng and analogue panax stipuleanatus |
| CN105063203A (en) * | 2015-08-07 | 2015-11-18 | 重庆出入境检验检疫局检验检疫技术中心 | Primers and probe for real-time fluorescent PCR detection of P. ginseng and detection method thereof |
| CN105624291A (en) * | 2016-01-19 | 2016-06-01 | 中国食品药品检定研究院 | Method for detecting whether Araliaceae plant components exist in sample and whether sample is adulterate |
| CN112522076A (en) * | 2020-12-08 | 2021-03-19 | 重庆海关技术中心 | Kit for identifying authenticity of ginseng |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103131781A (en) * | 2013-02-28 | 2013-06-05 | 中国科学院成都生物研究所 | PCR detection method of DNA of biological counterfeit drug in Chinese patent drug |
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| CN103173563A (en) * | 2013-04-15 | 2013-06-26 | 云南农业大学 | PCR-RFLP (Polymerase chain reaction-restriction fragment length polymorphism) method for quickly identifying panax notoginseng and analogue panax stipuleanatus |
| CN105063203A (en) * | 2015-08-07 | 2015-11-18 | 重庆出入境检验检疫局检验检疫技术中心 | Primers and probe for real-time fluorescent PCR detection of P. ginseng and detection method thereof |
| CN105624291A (en) * | 2016-01-19 | 2016-06-01 | 中国食品药品检定研究院 | Method for detecting whether Araliaceae plant components exist in sample and whether sample is adulterate |
| CN105624291B (en) * | 2016-01-19 | 2019-01-11 | 中国食品药品检定研究院 | It whether there is Araliaceae ingredient in test sample and whether mix pseudo- method |
| CN112522076A (en) * | 2020-12-08 | 2021-03-19 | 重庆海关技术中心 | Kit for identifying authenticity of ginseng |
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