CN110106277B - Kit and method for identifying radix glehniae and platycodon grandiflorum - Google Patents
Kit and method for identifying radix glehniae and platycodon grandiflorum Download PDFInfo
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- CN110106277B CN110106277B CN201910389098.7A CN201910389098A CN110106277B CN 110106277 B CN110106277 B CN 110106277B CN 201910389098 A CN201910389098 A CN 201910389098A CN 110106277 B CN110106277 B CN 110106277B
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Abstract
The invention relates to the technical field of traditional Chinese medicine identification, in particular to a kit and a method for identifying glehnia root and platycodon root. The kit comprises a glehnia root probe and a platycodon root probe, wherein the probes are connected with fluorescent labels. The thermodynamic efficiency of the combination of the probes and the templates is improved, so that each probe can be better combined with the DNA of the corresponding traditional Chinese medicine, but not combined with the DNA of the other two, the repeatability is good, the false positive rate is low, and the identification of the glehnia root, the platycodon root and the pseudo product of the glehnia root can be carried out through fluorescence detection after hybridization with the DNA, and the operation is simple, convenient and quick, and the cost is low.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine identification, in particular to a kit and a method for identifying glehnia root and platycodon root.
Background
Radix glehniae is a common traditional Chinese medicine, is the dry root of corallodiscus of Umbelliferae, and has the effects of nourishing yin, clearing lung-heat, eliminating phlegm, relieving cough, benefiting stomach and promoting fluid production; radix Platycodi is dry root of radix Platycodi of Campanulaceae, and has effects of dispersing lung qi, eliminating phlegm, relieving sore throat, and expelling pus. Because the price of the glehnia root and the platycodon root is higher, the appearance of the glehnia root and the platycodon root is similar to that of the camouflage, so that people in the market replace the glehnia root and the platycodon root with the camouflage. The traditional Chinese medicine decoction pieces can not be replaced at will in the use process, and the counterfeit products seriously affect the safety and the curative effect of the people in using the medicine. On the other hand, the situation that the radix glehniae and the genuine platycodon root are unintentionally confused exists in the actual use or purchasing process of the medicinal materials, so that potential safety hazards are brought to the medication. And when the glehnia root, the platycodon grandiflorum and the camouflage are processed into powder, the powder is not easy to distinguish. Therefore, the accurate identification of the glehnia root, the platycodon grandiflorum and the camouflage has important significance for the traditional Chinese medicine industry and the health of people. At present, conventional identification methods of traditional Chinese medicines comprise basic source identification, character identification, microscopic identification, physicochemical identification and the like, but have certain limitations on the processed traditional Chinese medicines.
In recent years, molecular identification methods based on DNA barcodes are rapidly developed, and become a new method for identifying traditional Chinese medicines after the traditional identification methods. Existing molecular identification techniques are mostly based on Polymerase Chain Reaction (PCR) and sequencing techniques: after the ITS2 sequence is obtained by PCR, sequencing and comparing sequence information are carried out on the PCR product, so that the genuine product and the counterfeit product can be distinguished. The molecular identification technology can ensure the reliability of test data, has high repeatability and reliable result, and is not influenced by the state of a sample and the harvesting processing when the molecular identification technology is used for identifying from the molecular level. But its economic cost and ease of use limit its potential for popularization due to the need for special and expensive sequencing instruments to sequence it (which typically need to be done by a sequencing company).
And then, fluorescent markers are inserted between bases in the PCR synthesis process by designing specific primers in a fluorescent marker mode so as to identify genuine products and counterfeit products. The method improves the defects of the prior art to a certain extent, but the fluorescence labeling cost of the full-sequence labeling is too high, the design and screening of the specific primers are time-consuming and labor-consuming, and the corresponding specific primers are difficult to obtain aiming at different traditional Chinese medicines to be identified, so that the popularization and the application of the technology are limited.
Disclosure of Invention
Aiming at the technical problems that the coastal glehnia root and the platycodon root and the coastal glehnia root and the camouflage celery can be confused and are not easy to distinguish in powder form, and the molecular identification method in the prior art is high in cost and not easy to design and screen specific primers, the invention provides a kit for identifying the coastal glehnia root and the platycodon root.
The invention also provides a method for identifying the glehnia root and the platycodon root.
The invention also provides application of the kit for identifying the glehnia littoralis and the platycodon grandiflorum in identifying the glehnia littoralis and the camouflage or identifying the platycodon grandiflorum and the camouflage.
In order to achieve the above purpose, the embodiment of the invention adopts the following technical scheme:
a kit for identifying radix glehniae and radix platycodonis comprises a radix glehniae probe and a radix platycodonis probe, wherein the radix glehniae probe and the radix platycodonis probe are connected with fluorescent markers; wherein the method comprises the steps of
The sequence of the glehnia littoralis probe is 5'-CCGTCGTAGGA-3' (shown as SEQ ID NO. 1);
the sequence of the platycodon grandiflorum probe is 5'-CTGCGAGGCAC-3' (shown as SEQ ID NO. 2).
The probe sequence in the kit is obtained by taking the sequence with larger difference in ITS2 difference sequences of radix glehniae, platycodon grandiflorum and camouflage as the basis and manually designing. Compared with the probes directly obtained according to the difference sequence, the thermodynamic efficiency of the probe and the template DNA is improved, so that each probe can be better combined with the DNA of the corresponding traditional Chinese medicine, but not combined with the DNA of the other two, the repeatability is good, and the false positive rate is low. After hybridization of the probe and DNA, the identification of the glehnia root, the platycodon root and the pseudo-product of the camouflage can be carried out through fluorescence detection, including the identification of decoction pieces or powder of the glehnia root and the platycodon root. The kit can obtain accurate identification results by using the probe, effectively reduces the single reaction cost, does not need a special detection instrument, greatly improves the usability of detection means, and can be widely applied to scientific research institutions, pharmacies, hospitals, processing and harvesting factories, traditional Chinese medicine suppliers and the like which relate to the identification of radix glehniae, platycodon grandiflorum and camouflage.
Preferably, the kit further comprises a plant ITS2 primer for amplifying genomic DNA of glehnia root, platycodon grandiflorum and camomile.
Preferably, the fluorescent label is attached to the 5' end of the probe. One probe is connected with only one fluorescent label, so that the use amount of fluorescent groups can be effectively reduced, and the cost is reduced.
Preferably, the fluorescent label is a Cy2 fluorescent label or an AlexaFluor488 fluorescent label.
The embodiment of the invention also provides a method for identifying the glehnia root and the platycodon root, and the kit for identifying the glehnia root and the platycodon root is used for identifying the glehnia root and the platycodon root.
Preferably, the specific operation is as follows: amplifying the genome DNA of the to-be-detected object, adding the glehnia littoralis probe or the platycodon grandiflorum probe for hybridization after the amplification is finished, and then carrying out electrophoresis and fluorescence detection.
Preferably, the hybridization method is as follows: adding the glehnia littoralis probe or the platycodon grandiflorum probe into the amplification primer, denaturing for 5min at 95 ℃, and annealing for 1min at 60 ℃. Under the preferable temperature and time conditions of denaturation and annealing, the probe can be better combined with DNA of the corresponding traditional Chinese medicine, but not combined with the other two, so that the three substances can be distinguished.
Preferably, the excitation wavelength of the fluorescence detection is 488nm.
Preferably, the electrophoresis condition is 1% agarose gel electrophoresis, and the voltage is 180V.
Preferably, the reaction system for amplifying the genomic DNA is: 100ng of genomic DNA or 10ng of plasmid DNA, 10 pmol/. Mu.L of ITS2 primer, 1X of DNA polymerase, and 50. Mu.L of DNA polymerase were filled with double distilled water; the reaction procedure is: 95 ℃ for 5min, and 1 cycle; 95 ℃ for 30s,54 ℃ for 30s and 72 ℃ for 30s, 25 cycles in total; 72 ℃ for 5min, and 1 cycle in total; the addition amount of the glehnia root probe or the platycodon root probe is 1 pmol/mu L.
The embodiment of the invention also provides application of the kit for identifying the glehnia littoralis and the platycodon grandiflorum in identifying the glehnia littoralis and the camouflage or identifying the platycodon grandiflorum and the camouflage. The kit can be used for distinguishing radix glehniae and radix platycodi from pseudo-camouflage.
Drawings
FIG. 1 shows the fluorescence detection results in example 2 of the present invention;
FIG. 2 shows the fluorescence detection results in example 3 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
The embodiment of the invention provides a kit for identifying glehnia root and platycodon root, which specifically comprises the following components:
(1) And (3) probe:
the sequence of the glehnia root probe is 5'-CCGTCGTAGGA-3';
the sequence of the balloonflower probe is 5'-CTGCGAGGCAC-3'.
(2) ITS2 primer; the forward primer sequence was 5'-ATGCGATACTTGGTGTGAAT-3' (shown as SEQ ID NO. 3) and the reverse primer sequence was 5'-GACGCTTCTCCAGACTACAAT-3' (shown as SEQ ID NO. 4).
(3) PCR reaction reagent: DNA polymerase 1×, double distilled water.
Example 2
The embodiment of the invention provides a method for identifying glehnia root and platycodon root, which uses the kit of the embodiment 1 for identification, and comprises the following steps:
(1) PCR reaction
Extracting genome DNA of the object to be detected, and carrying out PCR reaction by taking the genome DNA as a template according to the following system and program:
the system comprises: the genomic DNA of the test sample was 100ng, ITS2 primer was 10 pmol/. Mu.L, and DNA polymerase was added thereto and 1X mixed, and the mixture was supplemented to 50. Mu.L with double distilled water.
The reaction procedure:
95 ℃ for 5min, and 1 cycle in total;
95 ℃ for 30s,54 ℃ for 30s and 72 ℃ for 30s, 25 cycles in total;
at 72℃for 5min, 1 cycle.
(2) In situ probe binding
To the amplification primers, 1 pmol/. Mu.L of the glehnia littoralis probe or the platycodon grandiflorum probe was added, denatured at 95℃for 5min, and annealed at 60℃for 1min.
(3) Electrophoresis detection
The annealed samples (180V) were checked by 1% agarose gel electrophoresis and, after 10min, were subjected to fluorescent examination at an excitation wavelength of 488nm. The test results are shown in FIG. 1, wherein the left graph in FIG. 1 shows the electrophoresis result of the addition of the glehnia root probe, and the right graph shows the electrophoresis result of the addition of the platycodon root probe. The samples of radix glehniae and radix platycodi have obvious fluorescent stripes, while the pseudo-product camomile has no fluorescent stripes.
Example 3
The embodiment of the invention provides a method for identifying glehnia root and platycodon root, which uses the kit of the embodiment 1 for identification, and comprises the following steps:
(1) PCR reaction
Extracting plasmid DNA of the object to be detected, and carrying out PCR reaction by taking the plasmid DNA as a template according to the following system and program:
the system comprises: plasmid DNA of the test article 10ng, ITS2 primer 10 pmol/. Mu.L, DNA polymerase 1 Xwas added and mixed, and the mixture was made up to 50. Mu.L with double distilled water.
The reaction procedure:
95 ℃ for 5min, and 1 cycle in total;
95 ℃ for 30s,54 ℃ for 30s and 72 ℃ for 30s, 25 cycles in total;
at 72℃for 5min, 1 cycle.
(2) In situ probe binding
To the amplification primers, 1 pmol/. Mu.L of the glehnia littoralis probe or the platycodon grandiflorum probe was added, denatured at 95℃for 5min, and annealed at 60℃for 1min.
(3) Electrophoresis detection
The annealed samples (180V) were checked by 1% agarose gel electrophoresis and, after 10min, were subjected to fluorescent examination at an excitation wavelength of 488nm. The test results are shown in FIG. 2, wherein the left graph in FIG. 2 shows the electrophoresis result of the addition of the glehnia root probe, and the right graph shows the electrophoresis result of the addition of the platycodon root probe. The radix glehniae and the radix platycodi samples respectively show obvious fluorescent stripes, while the pseudo-product camomile does not show the fluorescent stripes.
The result shows that the kit provided by the invention has the advantages of simple and quick operation and low cost in the aspect of identifying the glehnia littoralis, the platycodon grandiflorum and the pseudo-product of the glehnia littoralis.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
SEQUENCE LISTING
<110> Hebei national institute of Chinese medicine
<120> kit for identifying radix Glehniae and radix Platycodi and identification method thereof
<130> 2019.05.05
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 11
<212> DNA
<213> glehnia littoralis Probe
<400> 1
ccgtcgtagg a
11
<210> 2
<211> 11
<212> DNA
<213> balloon flower Probe
<400> 2
ctgcgaggca c
11
<210> 3
<211> 20
<212> DNA
<213> ITS2 Forward primer
<400> 3
atgcgatact tggtgtgaat
20
<210> 4
<211> 21
<212> DNA
<213> ITS2 reverse primer
<400> 4
gacgcttctc cagactacaa t
21
Claims (9)
1. The kit for identifying the glehnia root and the platycodon root is characterized by being used for identifying the glehnia root and the camouflage or identifying the platycodon root and the camouflage; the kit comprises a glehnia root probe and a platycodon root probe, wherein the glehnia root probe and the platycodon root probe are connected with fluorescent markers; wherein the method comprises the steps of
The sequence of the glehnia littoralis probe is 5'-CCGTCGTAGGA-3';
the sequence of the platycodon grandiflorum probe is 5'-CTGCGAGGCAC-3'.
2. The kit for identifying glehnia root and platycodon root of claim 1, further comprising a plant ITS2 primer.
3. The kit for identifying glehnia root and platycodon root of claim 1, wherein the fluorescent label is attached to the 5' end of the probe.
4. The kit for identifying glehnia root and platycodon root according to claim 3, wherein the fluorescent label is a Cy2 fluorescent label or an AlexaFluor488 fluorescent label.
5. A method for identifying glehnia root and platycodon root, which is characterized in that the kit for identifying glehnia root and platycodon root according to any one of claims 1 to 4 is used for identifying glehnia root and platycodon root.
6. The method for identifying glehnia root and platycodon root according to claim 5, which is characterized by comprising the following specific operations: amplifying the genome DNA of the to-be-detected object, adding the glehnia littoralis probe or the platycodon grandiflorum probe for hybridization after the amplification is finished, and then carrying out electrophoresis and fluorescence detection.
7. The method for identifying glehnia root and platycodon root of claim 6, wherein the hybridization method is as follows: adding the glehnia littoralis probe or the platycodon grandiflorum probe into the amplification primer, denaturing for 5min at 95 ℃, and annealing for 1min at 60 ℃.
8. The method for identifying glehnia root and platycodon root according to claim 6, wherein the excitation wavelength of the fluorescence detection is 488nm; and/or
The electrophoresis condition is 1% agarose gel electrophoresis, and the voltage is 180V.
9. The method for identifying glehnia root and platycodon root according to claim 6, wherein a reaction system for amplifying the genomic DNA is as follows: 100ng of genomic DNA or 10ng of plasmid DNA, 10 pmol/. Mu.L of ITS2 primer, 1X of DNA polymerase, and 50. Mu.L of DNA polymerase were filled with double distilled water;
the reaction procedure is: 95 ℃ for 5min, and 1 cycle; 95 ℃ for 30s,54 ℃ for 30s and 72 ℃ for 30s, 25 cycles in total; 72 ℃ for 5min, and 1 cycle in total;
the addition amount of the glehnia root probe or the platycodon root probe is 1 pmol/mu L.
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| CN201910389098.7A CN110106277B (en) | 2019-05-10 | 2019-05-10 | Kit and method for identifying radix glehniae and platycodon grandiflorum |
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| CN110106277B true CN110106277B (en) | 2023-04-28 |
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2019
- 2019-05-10 CN CN201910389098.7A patent/CN110106277B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| Authentication of commercial processed Glehniae Radix (Beishashen) by DNA barcodes;Xunzhi Zhu等;《Chin Med》;20151130;第10卷(第35期);第1-9页 * |
| 北沙参及易混品的ITS2分子鉴定;李忠祥等;《沈阳药科大学学报》;20131031;第30卷(第10期);第803页摘要，右栏第1段 * |
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