CN115976247A - Primer, kit and identification method for nondestructively identifying American ginseng and ginseng - Google Patents
Primer, kit and identification method for nondestructively identifying American ginseng and ginseng Download PDFInfo
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Abstract
The invention discloses a primer, a kit and an identification method for nondestructively identifying American ginseng and ginseng, and mainly relates to the technical field of medicinal plant species identification. The primer comprises a nucleotide sequence shown in a sequence table SEQ ID No:1 and nucleotide sequences of the forward primer PF and the forward primer PF are shown in a sequence table SEQ ID No:2, and a reverse primer PR. The invention has the beneficial effects that: the ginseng and the American ginseng are identified by using a pair of primers, a reaction system is not required to be optimized, strict PCR reaction conditions are not required, and the ginseng and the American ginseng can be obviously distinguished through a characteristic peak of a melting curve or a Tm value of an amplification product, so that the aim of screening and identifying the ginseng and the American ginseng in fields quickly, nondestructively and in high throughput can be fulfilled.
Description
Technical Field
The invention relates to the technical field of medicinal plant species identification, in particular to a primer, a kit and an identification method for nondestructively identifying American ginseng and ginseng.
Background
The American ginseng and the ginseng are perennial herb plants of Araliaceae, and are two most widely applied medicinal plants in the ginseng genus. The Weihaiwenden in Shandong is the main production area of American ginseng in China, and the planting area of the American ginseng is more than 6 ten thousand mu. Ginseng is distributed in eastern parts of Liaoning, eastern halves of Jilin and eastern parts of Heilongjiang in China, and the cultivation area and yield account for about 70% of the world, wherein mountain ginseng and garden ginseng under the forest are two main types of ginseng at present. Because the plant characters of the ginseng and the American ginseng are similar, the production seeds have certain mixing, and the American ginseng or the ginseng in the field has a certain doping phenomenon. Although the plant forms and chemical components of ginseng and American ginseng have certain similarity, the ginseng and American ginseng have different pharmacological effects due to different types and contents of saponin. The pharmacopoeia of the people's republic of China describes that the medicine properties and the efficacies of the Chinese medicinal herbs have significant differences: the American ginseng is cool in nature, has the effects of tonifying qi and yin, clearing heat and promoting fluid production; ginseng, being warm in nature, has the effects of invigorating primordial qi, restoring pulse, relieving depletion, invigorating spleen, benefiting lung, promoting fluid production, and tranquilizing mind. The American ginseng and the ginseng cannot be used in a mixed way, so that the development of a method for rapidly and nondestructively identifying the American ginseng and the ginseng in the field plays a vital role in protecting the quality of the ginseng and the American ginseng products and guaranteeing the public health.
The traditional methods for identifying ginseng and American ginseng comprise a chemical method, a spectroscopic method and the like, but the methods need to extract and separate characteristic compounds of a sample, damage the sample per se during identification, and are not suitable for rapid screening and identification in fields.
In recent years, a series of DNA molecular labeling technologies, such as DNA barcodes, multiple site-specific PCR, etc., have also been developed for the identification of ginseng and american ginseng. When the DNA bar code is used for identification, the amplification, sequencing and cluster analysis of the bar code are required to be carried out on a sample to be detected every time; the multiple site-specific PCR is to design two or more pairs of primers by using multiple SNP sites of ginseng and American ginseng to amplify different target gene segments for multiple PCR identification. However, species-specific primers need to additionally introduce mismatched bases, and the temperature and primer concentration of a PCR reaction system need to be optimized to ensure the accuracy of the identification result. Due to the limitations of the above methods, it cannot be used for rapid identification and screening of large-scale American ginseng and ginseng in the field.
Disclosure of Invention
The invention aims to provide a primer, a kit and an identification method for nondestructively identifying American ginseng and ginseng, which can achieve the aim of rapidly, nondestructively and high-throughput screening and identifying ginseng and American ginseng in fields.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a primer for nondestructively identifying American ginseng and ginseng comprises a nucleotide sequence shown in a sequence table SEQ ID No:1 and the nucleotide sequence of the forward primer PF and the nucleotide sequence are shown in a sequence table SEQ ID No:2, reverse primer PR.
The forward primer PF is obtained from a front side flanking sequence of a specific insertion sequence based on ginseng, the reverse primer PR is obtained from a rear side flanking sequence of the specific insertion sequence based on ginseng, the length of the specific insertion sequence of ginseng is 365bp, and the nucleotide sequence of the specific insertion sequence is shown in a sequence table SEQ ID No:5, respectively.
As another aspect of the present invention, a kit, wherein the primers used in the kit are the forward primer PF and the reverse primer PR according to claim 1 or 2.
The kit comprises 1 mu L of primer PF,1 mu L of primer PR,2 mu L of sterilized ultrapure water and 5 mu L of SYBR Green premix, wherein the SYBR Green premix is prepared by passing 1 mu L of 10 multiplied by EasyTaq buffer,0.8 mu L of 2.5mM dNTPs,0.2 mu L of EasyTaq DNA polymerase,0.5 mu L of SYBR Green I and 2.5 mu. L H 2 And O, and the kit is used for identifying 1 mu L of DNA samples to be detected.
As another aspect of the present invention, an identification method, using the primers as defined in claim 1, performing PCR amplification on a DNA sample to be tested, wherein if the Tm value of the characteristic peak of the melting curve of the amplified product is 80.5 ℃, the sample is Panax ginseng, and if the Tm value of the characteristic peak is 76.5 ℃, the sample is Panax quinquefolium.
The adopted 10 muL fluorescent quantitative PCR reaction system is as follows: 1 mu L of genome DNA of a sample to be detected, 1 mu L of primer PF,1 mu L of primer PR,2 mu L of sterilized ultrapure water and 5 mu L of SYBR Green premix, wherein the SYBR Green premix passes through 1 mu L of 10×EasyTaq buffer，0.8μL 2.5mM dNTPs，0.2μL EasyTaq DNA polymerase,0.5μL SYBR Green I,2.5μL H 2 And mixing with O.
The fluorescent quantitative PCR reaction parameters are as follows: pre-denaturation at 95 ℃ for 15min; then, 40 cycles of 95 ℃ for 10s,58 ℃ for 10s and 72 ℃ for 20s are carried out; after the amplification cycle is finished, the temperature is reduced to 65 ℃, and then the temperature is raised to 95 ℃ to denature DNA products.
The method for extracting the DNA of the sample to be detected comprises the following steps: placing fresh leaves of a target plant into a 1.5mL eppendorf tube, adding 50 mu L of 0.5mol/L NaOH solution, adding sterilized steel balls, then carrying out vibration grinding for 30s at normal temperature by using a high-throughput tissue grinder, transferring 5 mu L of mixed solution into 295 mu L of Tris-HCl buffer solution, wherein the pH value of the buffer solution is 8.0, and blowing and uniformly mixing by using a liquid transferring gun to obtain a DNA sample.
Compared with the prior art, the invention has the beneficial effects that:
based on the primer, the genome DNA of a sample to be detected is subjected to fluorescent quantitative PCR amplification, and as the sequence of a target product of ginseng is 375bp longer than that of an American ginseng target product, and the Tm value of a ginseng amplification product is higher than that of an American ginseng amplification product, the characteristic peaks of the melting curves of the ginseng and the American ginseng are different, so that the ginseng and the American ginseng can be obviously distinguished according to the characteristic peak of the melting curve or the Tm value of the amplification product.
By the technology, the aim of quickly and accurately identifying the field American ginseng and ginseng plants can be fulfilled. Although the technologies such as DNA bar codes, multi-site specific PCR and the like can also identify the ginseng and the American ginseng, the DNA bar codes need to amplify, sequence and cluster analyze a sample to be detected each time; the multiple site-specific PCR requires two or more pairs of primers to amplify different target gene fragments, and the temperature and primer concentration of a PCR reaction system need to be optimized to ensure the reliability and accuracy of the identification result. Therefore, the invention has the creativity that only one pair of primers is used for identifying the ginseng and the American ginseng, and the reaction system is not required to be optimized, and the strict PCR reaction condition is not required. In addition, the method has low requirement on the purity of the DNA of the sample to be detected, and can extract the DNA of a small amount of ginseng or American ginseng leaves by adopting a simple method without damaging ginseng or American ginseng roots. The result can obviously distinguish the ginseng from the American ginseng through the characteristic peak of a melting curve or the Tm value of an amplification product, thereby achieving the aim of quickly and nondestructively identifying the American ginseng and the ginseng in the field.
Drawings
FIG. 1 is a relative position diagram of primers dedicated for identifying Panax quinquefolium and Panax ginseng.
FIG. 2 is a gel electrophoresis image of the general PCR identification results of Panax quinquefolium and Panax ginseng.
FIG. 3 is a melting curve diagram of the fluorescent quantitative PCR identification results of Panax quinquefolium and Panax ginseng.
FIG. 4 shows the operation of simple DNA extraction.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and these equivalents also fall within the scope of the present application.
The methods used in the examples are conventional methods unless otherwise specified, and the instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like known in the art and commercially available in the normal market unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art. The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. All the primer synthesis and sequence determination work were completed by Beijing Liu-He Hua Dageney science and technology Co.
The invention is based on a specific sequence with the length of 365bp which is found in CYP450 gene of ginseng by the inventor. The present invention is described in detail below with reference to examples for identifying ginseng and American ginseng.
Example 1: common PCR identification of American ginseng and ginseng
1. PCR special primer designed for identifying American ginseng and ginseng
1. Amplification and alignment of the Gene sequences of Panax quinquefolium and Panax ginseng CYP450
1.1 extraction of genomic DNA of Panax quinquefolium and Panax ginseng
Multiple American ginseng and ginseng leaves were collected from different production areas, washed with sterile water, ground into fine powder with liquid nitrogen in a mortar, and then extracted with genomic DNA of American ginseng and ginseng samples using a Plant genomic DNA extraction Kit (DNeasy Plant Mini Kit, QIAGEN) according to the instructions.
Table 1 American Ginseng and Ginseng samples
1.2PCR amplification of CYP450 Gene sequences of Panax quinquefolium and Panax ginseng, respectively
20 μ L of PCR reaction system: 1 μ L (10 ng) of genomic DNA of Panax quinquefolium or Panax ginseng, 2 μ L (0.5 μ M) of primer cypF (5'-GATAAAGGAGTTGATGTGTGAGC-3'), 2 μ L (0.5 μ M) of primer cypR (5'-CAGGAAGGACAACACCCGAC-3'), 5 μ L of sterilized ultrapure water, 10 μ L of 2 × Easy Taq Supermix DNA polymerase (formulation: 2 μ L of 10 × Easy Taq buffer,1.60 μ L of 2.5mM dNTPs,0.4 μ L of Easy Taq DNA polymerase,6 μ L H 2 O)。
The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min; then, 35 cycles of reaction are carried out at 94 ℃ 30s,58 ℃ 30s and 72 ℃ 90 s; finally, the reaction is ended by extension at 72 ℃ for 7min and heat preservation at 4 ℃.
1.3 sequence alignment
And (3) directly sequencing the PCR amplification product, and splicing the DNA sequencing result by utilizing SeqMan software. The sequences were aligned using ClustalW Omega software and the results are shown in FIG. 1. The amplification sequence of the American ginseng is shown as SEQ ID No:3, the amplified sequence of the ginseng is shown as SEQ ID No:4, the insertion sequence of the ginseng with the length of 365bp is shown as SEQ ID No:5, respectively.
2. PCR special primer designed for identifying American ginseng and ginseng
According to the specific insertion sequence of the ginseng found in the sequence alignment, a forward primer PF is designed at the 117 th to 138 th bases of the sequence table 1, and the sequence is 5'-ATTAATTGAAAAACTTGATGAC-3' (SEQ ID No:1 in the sequence table). Reverse primer PR is designed at the base positions 567-586, and the sequence is 5'-TGACCTTGATCAGTACCAAT-3' (SEQ ID No:2 in the sequence table).
2. PCR identification of American ginseng and ginseng
The genomic DNA of the American ginseng and the genome DNA of the ginseng are respectively used as templates, and simple PCR special primers PF and PR for identifying the American ginseng and the ginseng are used for PCR amplification.
The 20 μ L multiplex PCR reaction was: mu.L (10 ng) of genomic DNA of a sample to be tested, 2. Mu.L (0.5. Mu.M) of primer PF, 2. Mu.L (0.5. Mu.M) of primer PR, 5. Mu.L of sterilized ultrapure water, 10. Mu.L of 2 XPpremium DNA polymerase (formulation: 2. Mu.L of 10 XPaEasyTaq buffer, 1.6. Mu.L of 2.5mM dNTPs, 0.4. Mu.L of EasyTaq DNA polymerase, 6. Mu. L H 2 O)). The general PCR reaction conditions were: pre-denaturation at 94 ℃ for 4min; then, 35 cycles of reaction are carried out at 94 ℃ 30s,58 ℃ 30s and 72 ℃ 60 s; finally, extension is carried out for 5min at 72 ℃, and the reaction is finished after heat preservation at 4 ℃.
The results of 1% agarose gel electrophoresis detection of 3. Mu.L of the multiplex PCR amplification products are shown in FIG. 2 (lane 1 is blank control, 2-7 are ginseng from different origins, 8-13 are American ginseng from different origins). As can be seen from FIG. 2, all the samples of Panax quinquefolium produced a 105bp DNA fragment amplified by the primers PF and PR. While the 470bp DNA fragment was amplified from the ginseng samples from different producing areas. Therefore, the primers and the conventional PCR technology can be used for easily distinguishing and identifying the American ginseng.
Example 2: rapid nondestructive identification of field American ginseng and ginseng
1. Simple extraction of field American ginseng and ginseng genome DNA
About 10mg of fresh leaves are put into 1.5mL of eppendorf, 50 μ L of 0.5mol/L NaOH solution is added, and after the sterilized steel balls are added, the fresh leaves are ground by a high-throughput tissue grinder for 30s under normal temperature. Transfer 5. Mu.L of the mixture into 295. Mu.L of Tris-HCl buffer (pH 8.0), and gently blow and mix the mixture with a pipette to obtain a simple extracted DNA sample (FIG. 3).
2. Fluorescent quantitative PCR identification of field American ginseng and ginseng
1 mu L of DNA samples of the American ginseng which are simply extracted are respectively used for fluorescent quantitative PCR identification. The reaction system of 10 μ L fluorescent quantitative PCR is: 1. Mu.L of genomic DNA of a sample to be tested, 1. Mu.L (0.5. Mu.M) of primer PF, 1. Mu.L (0.5. Mu.M) of primer PR, 2. Mu.L of sterilized ultrapure water, 5. Mu.L of SYBR Green premix (formulation: 1. Mu.L of 10 XSeTaq buffer, 0.8. Mu.L of 2.5mM dNTPs, 0.2. Mu.L of EasyTaq DNA polymerase, 0.5. Mu.L of SYBR Green I, 2.5. Mu. L H 2 O)。
The fluorescent quantitative PCR reaction parameters are as follows: pre-denaturation at 95 ℃ for 15min; then, 40 cycles of 95 ℃ for 10s,58 ℃ for 10s and 72 ℃ for 20s are carried out; after the amplification cycle is finished, the temperature is reduced to 65 ℃, and then the temperature is increased to 95 ℃ to denature the DNA product. FIG. 4 shows the melting curve of the fluorescent quantitative PCR. The Tm values of all ginseng samples are 80.5 ℃, the Tm values of all American ginseng samples are 76.5 ℃, and the ginseng and the American ginseng can be obviously distinguished and screened according to the characteristic peak of a melting curve or the Tm value of an amplification product.
Claims (8)
1. A primer for nondestructively identifying American ginseng and ginseng is characterized by comprising a nucleotide sequence shown in a sequence table SEQ ID No:1 and nucleotide sequences of the forward primer PF and the forward primer PF are shown in a sequence table SEQ ID No:2, reverse primer PR.
2. The primer for nondestructive identification of American ginseng and ginseng according to claim 1, wherein the forward primer PF is obtained based on a front flanking sequence of a specific insertion sequence of ginseng, the reverse primer PR is obtained based on a rear flanking sequence of the specific insertion sequence of ginseng, the length of the specific insertion sequence of ginseng is 365bp, and the nucleotide sequence of the specific insertion sequence of ginseng is shown in a sequence table SEQ ID No:5, respectively.
3. A kit, characterized in that the primers used in the kit are the forward primer PF and the reverse primer PR according to claim 1 or 2.
4. The kit of claim 3, wherein the kit comprises 1 μ L of PF primer, 1 μ L of PR primer, 2 μ L of sterilized ultrapure water, 5 μ L of SYBR Green premix prepared by passing 1 μ L of 10 × EasyTaq buffer,0.8 μ L of 2.5mM dNTPs,0.2 μ L of EasyTaq DNA polymerase,0.5 μ L of SYBR Green I primer, 2.5 μ L H 2 And O, and the kit is used for identifying 1 mu L of DNA samples to be detected.
5. An identification method, characterized in that, the primer of claim 1 is used to perform PCR amplification on a DNA sample to be detected, if the Tm value of the characteristic peak of the melting curve of the amplification product is 80.5 ℃, the sample is ginseng, and if the Tm value of the characteristic peak is 76.5 ℃, the sample is American ginseng.
6. An identification method according to claim 5, wherein the 10 μ L fluorescent quantitative PCR reaction system is: 1 mu L of genome DNA of a sample to be detected, 1 mu L of primer PF,1 mu L of primer PR,2 mu L of sterilized ultrapure water, 5 mu L of SYBR Green premix which is prepared by mixing 1 mu L of 10 XEasyTaq buffer,0.8 mu L of 2.5mM dNTPs,0.2 mu L of EasyTaq DNA polymerase,0.5 mu L of SYBR Green I,2.5 mu L L H 2 And mixing the components O and O.
7. An identification method according to claim 5, wherein the fluorescent quantitative PCR reaction parameters are: pre-denaturation at 95 ℃ for 15min; then, 40 cycles of 95 ℃ for 10s,58 ℃ for 10s and 72 ℃ for 20s are carried out; after the amplification cycle is finished, the temperature is reduced to 65 ℃, and then the temperature is raised to 95 ℃ to denature DNA products.
8. An identification method according to claim 5, wherein said method for extracting DNA from a sample to be tested comprises: placing fresh leaves of a target plant into a 1.5mL eppendorf tube, adding 50 mu L of 0.5mol/L NaOH solution, adding sterilized steel balls, then carrying out vibration grinding for 30s at normal temperature by using a high-throughput tissue grinder, transferring 5 mu L of mixed solution into 295 mu L of Tris-HCl buffer solution, wherein the pH value of the buffer solution is 8.0, and blowing and uniformly mixing by using a liquid transferring gun to obtain a DNA sample.
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