CN102827940B - 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 ginseng in Chinese patent medicine.
Background technology
The quality control of traditional Chinese patent medicine is only limitted to the assay to the discriminating on property of drug and compound.Chinese patent medicine made by Chinese medicinal materials, and crude drug proterties of itself when several formulations technique makes Chinese patent medicine has been difficult to distinguish, and the complicated component of Chinese medicine own, Herba indigoferae Pseudotinctoriae is with regard to tens even hundreds of compounds, a Chinese patent medicine be made up of plurality of Chinese, wherein chemical composition is just more, only select the qualification of limited several chemical compositions to control the quality of Chinese patent medicine at present, just for Chinese patent medicine fake producer provides very large space, for certain expensive Chinese medicinal materials, such as ginseng, ginseng crude drug is not added in production process, only add the compound being listed in test item, or pass a fake product off as a genuine one with other medicinal materials containing same compound, present method is by the amplification of PCR, powder is beaten to passing through, extract, Chinese patent medicine after filtration still can detect the DNA of wherein ginseng, overcome the deficiencies in the prior art, can be easy, fast, judge whether use ginseng in Chinese patent medicine exactly, ensure drug quality.
Summary of the invention
The object of this invention is to provide a kind of method that PCR detects Ginseng DNA in Chinese patent medicine.
Aforesaid method of the present invention utilizes polymerase chain reaction (PCR) to increase in Chinese patent medicine the specific DNA of ginseng to detect the Ginseng DNA in Chinese patent medicine.Wherein detected Chinese patent medicine is be Panax ginseng C.A.Mey., its another name or Chinese patent medicine of its processed product containing Classification system.
Aforesaid method of the present invention comprises the following steps:
1) extraction of Ginseng DNA in Chinese patent medicine;
2) PCR reaction;
3) pcr amplification product analysis.
In aforesaid method, step 1) in adopt improved method of CTAB to extract DNA for Chinese patent medicine medicinal material with the formulation that medicinal powder is used as medicine; The method of ethyl alcohol recrystallization is adopted to extract for Chinese patent medicine medicinal material with the formulation that extracting solution is used as medicine.
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 aforesaid method, step 2) in regular-PCR is adopted for the sample that DNA content is higher, the sample lower for DNA content adopts nest-type PRC.
The Auele Specific Primer that the forward primer of wherein said common PCR reaction and reverse primer are is stencil design with ginseng ITS2 sequence area, its object band can at 80-224bp; Described nest-type PRC carries out first round PCR and second successively to total genomic dna and takes turns PCR twice amplification, and the DNA fragmentation of first round pcr amplification product can at 700-1500bp, and second to take turns PCR identical with regular-PCR.
Particularly, the method that a kind of PCR provided by the invention detects Ginseng DNA in Chinese patent medicine comprises the following steps:
1. the extraction of Ginseng DNA in Chinese patent medicine:
Suitable extracting method can be selected according to different formulations.
The medicine that medicinal powder is used as medicine comprises the formulations such as tablet, capsule, pill and adopts improved method of CTAB to extract.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, adds the CTAB Extraction buffer 700 μ l of 65 DEG C of preheatings, mixing of fully vibrating, 65 DEG C of temperature bath 2h; 2. sample adds chloroform-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, in the EP pipe that transfer supernatant liquor to is new; 4. add the Virahol of equal-volume-20 DEG C of precoolings, mixing, place 30 minutes for 20 DEG C; 5. 10,000r.min
-1centrifugal 15 minutes, abandon supernatant liquor; 6. precipitation is respectively washed once with 70% ethanol, dehydrated alcohol, 10,000r.min
-1centrifugal 3 minutes, abandon supernatant liquor, room temperature volatilized ethanol, aseptic high purity water dissolving DNA, for subsequent use.Described CTAB Extraction buffer contains: 2%CTAB (cetyl trimethylammonium bromide), 100mM Tris (pH 8.0), 20mMEDTA (pH 8.0), 2.5M NaCl.
What extracting solution was used as medicine comprises the method extraction that oral liquid, injection liquid etc. adopt ethyl alcohol recrystallization.1. 10mL liquid adds 1mL 3M sodium-acetate (pH=5.2), 30mL dehydrated alcohol, 30 μ l glycogens (glycogen), places 1.5h for-20 DEG C.2. 14,000r.min
-1centrifugal 10 minutes, abandon supernatant.3. 70% washing with alcohol, 16,000r.min
-1centrifugal 5 minutes, abandon supernatant.4. ethanol is volatilized, aseptic high purity water dissolving DNA, for subsequent use.
2. common PCR reaction system and condition:
10 × Taq buffer 5 μ l; Taq enzyme 5 units;
Forward primer 1 μM; Reverse primer 1 μM;
DNTPs 0.2mM; DNA extraction liquid 1 μ l.
95 DEG C are unwind 3 minutes; 94 DEG C of sex change 30 seconds, 55 DEG C of renaturation 30 seconds, 72 DEG C extend 30 seconds, circulate 30 times; 72 DEG C extend 5 minutes, obtain amplification sample.
3. nest-type PRC reaction system and condition:
First round PCR:
10 × Taq buffer 5 μ l; Taq enzyme 5 units;
Forward primer 0.25 μM; Reverse primer 0.25 μM;
DNTPs 0.2mM; DNA extraction liquid 5 μ l.
95 DEG C are unwind 3 minutes; 94 DEG C of sex change 30 seconds, 60 DEG C of renaturation 30 seconds, 72 DEG C extend 30 seconds, circulate 25 times; 72 DEG C extend 5 minutes, obtain amplification sample.
Second takes turns PCR operates with regular-PCR.
4.PCR amplified production is analyzed:
Get the sample after 5 μ l amplifications, mix with 1 μ l, 6 times of DNA sample-loading buffers, agarose gel containing Gelview nucleic acid dye carries out electrophoresis and colour developing, observes under ultraviolet transmissive lamp, what sample well contained the band of an entry is the positive containing ginseng or pollution.
The principle of the invention is: the DNA that can be extracted plant in Chinese patent medicine by the method for CTAB method or ethyl alcohol recrystallization, containing polymerase chain reaction reagent in PCR reaction reagent pipe, special amplification can be carried out to the DNA of ginseng in Reagent Tube, due to primer of the present invention by ginseng is peculiar, therefore, if containing ginseng in sample, so its DNA specific fragment is after amplification, agarose gel containing Gelview nucleic acid dye carries out electrophoresis and colour developing, UV-light detects the object band that just can detect certain length fragment, if there is no ginseng, then can not increase the object band of same long fragment.
The present invention has the advantage being obviously better than prior art, and its major advantage comprises:
Novelty.Present method is that the method first PCR being detected DNA is applied in the discriminating of the true or false of Chinese patent medicine.
Rapidity.Adopt the inventive method to detect very quick, within 4-6 hour, can detected result be learnt.
Susceptibility.Due to the stability of DNA itself, by being still totally disrupted after the processing of various preparation process, then pass through the amplification of PCR (polymerase chain reaction) exponential type, and nest-type PRC can detect the DNA of denier as supplementary means.
Accompanying drawing explanation
Fig. 1 is specific embodiment 1 detected result agarose gel figure; Wherein A is negative control, and B is tablet, and C is positive control, and D is Marker.
Fig. 2 is specific embodiment 2 detected result agarose gel figure; A is negative control, and B is injection, and C is positive control, and D is Marker.
Embodiment
Below in conjunction with accompanying drawing, further illustrate the present invention by example.One skilled in the art will understand that these examples are only for illustration of the present invention, and be not used in and limit the scope of the invention.
The DNA of ginseng in embodiment 1, detection ginseng-astragalus sheet
Prepare Ginseng DNA according to following formula and extract reagent
CTAB Extraction buffer: 2%CTAB (cetyl trimethylammonium bromide), 100mMTris (pH 8.0), 20mM EDTA (pH 8.0), 2.5M NaCl.Virahol, chloroform, dehydrated alcohol, 70% ethanol, aqua sterilisa.
Detect according to follow procedure
1. the extraction of ginseng 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, adds the CTAB Extraction buffer 700 μ l of 65 DEG C of preheatings, mixing of fully vibrating, 65 DEG C of temperature bath 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, in the EP pipe that transfer supernatant liquor to is new; 4. add the Virahol of equal-volume-20 DEG C of precoolings, mixing, place 30 minutes for 20 DEG C; 5. 10,000r.min
-1centrifugal 15 minutes, abandon supernatant liquor; 6. precipitation is respectively washed once with 70% ethanol, dehydrated alcohol, 10,000r.min
-1centrifugal 3 minutes, abandon supernatant liquor, room temperature volatilized ethanol, aseptic high water dissolution DNA, for subsequent use.
2. the extraction (with control medicinal material DNA extraction method) of DNA of plants in ginseng-astragalus sheet
3.PCR increases
Forward primer: GGGGCGGATAATGGC
Reverse primer: ACGACATGAGAAGAGGGC
PCR reaction system:
10 × Taq buffer 5 μ l; Taq enzyme 5 units;
Forward primer 1 μM; Reverse primer 1 μM;
DNTPs 0.2mM; DNA extraction liquid 1 μ l;
System is supplied 50 μ l by ultrapure aqua sterilisa.
And control medicinal material DNA extraction liquid and ultrapure aqua sterilisa are set simultaneously respectively as positive control, negative control.
PCR reaction conditions: 95 DEG C are unwind 3 minutes; 94 DEG C of sex change 30 seconds, 55 DEG C of renaturation 30 seconds, 72 DEG C extend 30 seconds, circulate 30 times; 72 DEG C extend 5 minutes, obtain amplification sample.
4.PCR amplified production is analyzed
Send order-checking company to check order ginseng control medicinal material PCR primer, sequence is correct.
Get the sample after 5 μ l amplifications, mix with 1 μ l, 6 times of DNA sample-loading buffers, agarose gel containing Gelview nucleic acid dye carries out electrophoresis and colour developing, observe under ultraviolet transmissive lamp, sample well contains one and to illustrate in this medicine with the object band of positive control same position and add ginseng crude drug.
Detected result agarose gel figure as shown in Figure 1.
The DNA of ginseng in embodiment 2, detection Shenmai injection
The preparation of positive control and same example 1. is set
Detect according to follow procedure:
1. the extraction of Ginseng DNA in Shenmai injection
1. 10mL injection liquid adds 1mL 3M sodium-acetate (pH=5.2), 30mL dehydrated alcohol, 30 μ l glycogens (glycogen), places 1.5h for-20 DEG C.2. 14,000r.min
-1centrifugal 10 minutes, abandon supernatant.3. 70% washing with alcohol, 16,000r.min
-1centrifugal 5 minutes, abandon supernatant.4. ethanol is volatilized, aseptic high water dissolution DNA, for 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
PCR reaction system:
10 × Taq buffer 5 μ l; Taq enzyme 5 units;
Forward primer 0.25 μM; Reverse primer 0.25 μM;
DNTPs 0.2mM; DNA extraction liquid 1 μ l;
System is supplied 50 μ l by ultrapure aqua sterilisa.
And control medicinal material DNA extraction liquid and ultrapure aqua sterilisa are set simultaneously respectively as positive control, negative control.PCR reaction conditions: 95 DEG C are unwind 3 minutes; 94 DEG C of sex change 30 seconds, 60 DEG C of renaturation 30 seconds, 72 DEG C extend 30 seconds, circulate 25 times; 72 DEG C extend 5 minutes, obtain amplification sample.
Second takes turns PCR
Forward primer: GGGGCGGATAATGGC
Reverse primer: ACGACATGAGAAGAGGGC
PCR reaction system:
10 × Taq buffer 5 μ l; Taq enzyme 5 units;
Forward primer 1 μM; Reverse primer 1 μM;
DNTPs 0.2mM; First round PCR primer 1 μ l;
System is supplied 50 μ l by ultrapure aqua sterilisa.
And get the negative control of first round moderate and positive control increases respectively as negative control, positive control simultaneously.
PCR reaction conditions: 95 DEG C are unwind 3 minutes; 94 DEG C of sex change 30 seconds, 55 DEG C of renaturation 30 seconds, 72 DEG C extend 30 seconds, circulate 30 times; 72 DEG C extend 5 minutes, obtain amplification sample.
3. nested PCR amplification and product analysis
Get the sample after 5 μ l amplifications, mix with 1 μ l, 6 times of DNA sample-loading buffers, agarose gel containing Gelview nucleic acid dye carries out electrophoresis and colour developing, observe under ultraviolet transmissive lamp, sample well contains one and to illustrate in this medicine with the object band of positive control same position and add ginseng crude drug.
Detected result agarose gel figure as shown in Figure 2.
Claims (1)
1. the PCR detection method of Ginseng DNA in Chinese patent medicine, it is characterized in that, described Chinese patent medicine is Shenmai injection; Described PCR detection method comprises the following steps: 1) extraction of Ginseng DNA in Shenmai injection; 2) PCR reaction; 3) pcr amplification product analysis; Wherein, step 1) in, adopt the method for ethyl alcohol recrystallization to extract; Step 2) in, the sample higher for DNA content adopts regular-PCR, and the sample lower for DNA content adopts nest-type PRC; The Auele Specific Primer that the forward primer of described common PCR reaction and reverse primer are is stencil design with ginseng ITS2 sequence area, forward primer is GGGGCGGATAATGGC, and reverse primer is ACGACATGAGAAGAGGGC; Described nest-type PRC carries out first round PCR and second successively to genomic dna and takes turns PCR twice amplification, the forward primer of first round PCR is CACACCGCCCGTCGCTCCTACCGA, reverse primer is ACTCGCCGTTACTAGG GGAA, and the second ginseng Auele Specific Primer of taking turns the primer of PCR used with above-mentioned regular-PCR is identical.
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| CN103131781B (en) * | 2013-02-28 | 2015-10-28 | 中国科学院成都生物研究所 | The PCR detection method of biological species adulterant DNA in Chinese patent medicine |
| 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 |
| 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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| CN1557968A (en) * | 2004-02-11 | 2004-12-29 | 复旦大学 | Molecular mark of wild mountain ginseng and discrimination method therefor |
| CN101654709A (en) * | 2008-08-18 | 2010-02-24 | 韩国农村振兴厅 | Method for using sts primer to identify ginseng species |
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