CN111676310A - Primer pair, method and application for identifying or assisting in identifying polygonatum cyrtonema - Google Patents
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- 102000053602 DNA Human genes 0.000 claims description 8
- 241000037826 Polygonatum kingianum Species 0.000 claims description 5
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 4
- 210000003763 chloroplast Anatomy 0.000 claims description 4
- 239000003147 molecular marker Substances 0.000 abstract description 6
- 241000037831 Polygonatum sibiricum Species 0.000 abstract description 5
- 238000012163 sequencing technique Methods 0.000 abstract description 5
- 238000012300 Sequence Analysis Methods 0.000 abstract description 4
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- 208000006083 Hypokinesia Diseases 0.000 description 1
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Abstract
The invention discloses a primer pair, a method and application for identifying or assisting in identifying polygonatum cyrtonema. Performing PCR amplification on the genomic DNA of a sample to be detected by using the primers; if the amplification product contains a DNA fragment of about 1500bp, the sample to be detected is or is selected as polygonatum cyrtonema; if the amplified product does not contain a DNA fragment of about 1500bp, the sample to be detected is the polygonatum cyrtonema which is not polygama. The invention relates to a method for detecting the specificity of a SNP molecular marker, which is characterized in that a polygonatum sibiricum sample for 3 pharmacopoeia containing all species of polygonatum sibiricum is sequenced and subjected to sequence analysis by sequencing and sequence analysis technologies, so that the SNP molecular marker only existing in the polygonatum sibiricum is found, and the method for detecting the specificity of the SNP molecular marker is established.
Description
Technical Field
The invention relates to a traditional Chinese medicine specific molecular marker technology, in particular to a primer pair, a method and application for identifying or assisting in identifying polygonatum cyrtonema.
Background
Polygonatum cyrtonema hua is one of the original plants of Polygonatum cyrtonema Hua recorded in the 2020 edition of Chinese pharmacopoeia, has the effects of invigorating qi, nourishing yin, strengthening spleen, moistening lung, tonifying kidney and the like, and is used for treating spleen and stomach qi deficiency, tiredness, hypodynamia, insufficiency of essence and blood and the like. The original herb recorded in famous medical records of this chapter carries Siberian solomonseal rhizome, which is sweet, mild and nontoxic in flavor. Has the effects of invigorating spleen and replenishing qi, dispelling wind and dampness, and calming five internal organs. Can be taken for a long time to lighten the body, prolong life, and avoid hunger. Therefore, the sealwort has been used as a large amount of tonifying medicinal materials, has great medicinal value and can also be used as a medicated diet. Polygonatum cyrtonema is mainly distributed in east China, China and south China, Sichuan China, Guizhou, Hunan, Hubei, Henan, Anhui, and the like.
The site-specific PCR identification technology has been successfully applied to identification of polygonatum cyrtonema and counterfeit products thereof, but effective identification of three polygonatum plants of polygonatum medicinal materials recorded in pharmacopoeia is not carried out, and SNP sites are mostly searched through a section of genome sequence and specific primers are designed for identification.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to rapidly identify polygonatum cyrtonema in three germplasms specified in pharmacopoeia polygonatum provides a primer pair, a method and application for identifying or assisting in identifying polygonatum cyrtonema.
The invention solves the technical problems through the following technical scheme, and the primer pair for identifying or assisting in identifying polygonatum cyrtonema of the invention consists of primers PCY1-F and PCY1-R, wherein the primers PCY1-F are (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in a sequence 2 in a sequence table;
(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;
the primer PCY1-R is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in a sequence 3 in a sequence table;
(a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3.
The primer pair is applied to identification or auxiliary identification of polygonatum cyrtonema.
The chloroplast genome of the medicinal polygonatum cyrtonema is a DNA molecule shown as a sequence 1 in a sequence table.
A method for identifying or assisting in identifying polygonatum cyrtonema includes the following steps:
(1) performing PCR amplification on the genomic DNA of a sample to be tested by using the primer according to claim 1;
(2) if the amplification product contains a DNA fragment of about 1500bp, the sample to be detected is or is selected as polygonatum cyrtonema; if the amplified product does not contain a DNA fragment of about 1500bp, the sample to be detected is the polygonatum cyrtonema which is not polygama.
The genotype of a specific SNP site in the genome DNA of a sample to be detected is a CT genotype, and the sample to be detected is polygonatum cyrtonema;
the genotype of a specific SNP site in the genome DNA of a sample to be detected is a non-CT genotype, and the sample to be detected is non-polygonatum cyrtonema.
The specific SNP site is the 153809 th nucleotide site from the 5' end in the sequence 1.
The DNA fragment of about 1500bp is 1507 bp.
The sample to be detected is Polygonatum cyrtonema, Polygonatum sibiricum and Polygonatum kingianum.
A kit for identifying or assisting in identifying polygonatum cyrtonema includes the primer pair.
The method is characterized in that a full-length sequence is obtained by sequencing a chloroplast genome of polygonatum cyrtonema, a data base of the full-length sequence of the chloroplast genome is obtained by sequencing polygonatum cyrtonema and polygonatum kingianum, a specific mutation site of polygonatum cyrtonema is found out by a clustalW comparison function, a specific primer is designed by utilizing an amplification retardation variation principle, and polygonatum cyrtonema in three germplasms specified by the polygonatum cyrtonema in pharmacopoeia is rapidly identified.
Compared with the prior art, the invention has the following advantages: the invention carries out sequencing and sequence analysis on rhizoma polygonati samples for 3 pharmacopoeias containing all species of polygonatum cyrtonema by sequencing and sequence analysis technologies, finds out an SNP molecular marker only existing in polygonatum cyrtonema, and establishes a specific detection method of the SNP marker.
Drawings
FIG. 1 is a schematic diagram of a double block specific primer design;
FIG. 2 shows the result of electrophoresis of PCR amplification products of a sample.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
The three polygonatum plants in the following examples are published and recorded in the 'Chinese pharmacopoeia' and 'Chinese plant record' of 2020 edition; from which the public can refer to references. All samples in the table accord with the relevant regulations under the terms of each medicinal material, such as Chinese pharmacopoeia, compendium of materia Medica, and the like. Through identification, the material objects of the medicines accord with the names, and the quality accords with the standard.
10 × Taq buffer, Taq DNA polymerase: takara, catalog number R001A.
Example 1
In this example, 3 polygonatum sibiricum samples including all species of polygonatum cyrtonema complex in table 1 were sequenced and sequence analyzed, and a single SNP molecular marker existing only in polygonatum cyrtonema was found on the genome sequence.
The SNP site and the nucleotides in the vicinity thereof are shown in SEQ ID NO. 1, wherein the 153809 th nucleotide is the SNP site and the 155360 th nucleotide is the SNP site. A pair of primers is designed for the SNP sites as shown in Table 1:
TABLE 1 primer pair information
The primer design scheme is shown in FIG. 1, and FIG. 1 is a design scheme of a double block specific primer. Wherein, the 3 'end of the forward primer is completely matched with 153809 th site of polygonatum cyrtonema, the 2 nd site from the last of the 3' end is introduced with mismatch C, the 3 'end of the reverse primer is completely matched with 155360 th site of polygonatum cyrtonema, and the 2 nd site from the last of the 3' end is introduced with mismatch G to enhance the specificity of the primers.
The method for identifying polygonatum cyrtonema is as follows:
(1) extracting the genome DNA of a sample to be detected;
(2) and (2) carrying out PCR amplification by using the genomic DNA obtained in the step (1) as a template and using the primer PCY1-F and the primer PCY1-R to obtain an amplification product.
Reaction system for PCR amplification (20 μ L): 2 μ L of 10 XTaq buffer, 2 μ L of 2.5mmol/L dNTPs, 0.5 μ L of primer PCY1-F, 0.5 μ L of primer PCY1-R, 0.2 μ L of Taq DNA polymerase, 1 μ L (1-100ng) of DNA template, and sterile distilled water to make up to 20 μ L. The primers PCY1-F and the primers PCY1-R are added into a PCR reaction system in a primer solution form, and the initial concentrations of the primers PCY1-F and the primers PCY1-R in the primer solution are both 10 mu M.
Reaction procedure for PCR amplification: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min, and 30 cycles; extension at 72 ℃ for 7 min.
(3) And (3) carrying out 1.0% agarose gel electrophoresis on the amplification product obtained in the step (2).
The judgment method comprises the following steps: if the amplification product contains a DNA fragment of about 1500bp, the sample to be detected is or is selected as polygonatum cyrtonema; if the amplified product does not contain the DNA fragment of about 1500bp, the sample to be detected is or is selected as non-polygonatum cyrtonema.
Example 2
Table 2 shows the information collected from three medicinal Polygonatum materials. All samples in the table accord with the relevant regulations under the terms of each medicinal material, such as Chinese pharmacopoeia, compendium of materia Medica, and the like. Through identification, the material objects of the medicines accord with the names, and the quality accords with the standard.
TABLE 2 rhizoma Polygonati sample information table
The test method established in example 1 was used to test the test samples of table 2. The electrophoresis results are shown in FIG. 2.
The results of specific PCR detection by the method of example 1 are consistent with the actual conditions, and the detection results all have polygonatum cyrtonema specific bands.
FIG. 2 shows the result of electrophoresis of PCR amplification products of a sample. Wherein M is DL2000 DNA molecular weight standard (the sizes of bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom respectively); lanes 1-4 polygonatum cyrtonema samples, 1: polygonatum cyrtonema (numbered 1 in Table 2); 2: polygonatum cyrtonema (numbered 2 in Table 2); 3: polygonatum cyrtonema (numbered 3 in Table 2); 4: polygonatum cyrtonema (numbered 4 in Table 2); lanes 5-8 are non-polygonatum cyrtonema samples, 5: sealwort (numbered 5 in table 2); 6: sealwort (numbered 6 in table 2); 7: polygonatum kingianum (numbered as 7 in table 2); 8: polygonatum kingianum (numbered as 8 in Table 2).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
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Claims (9)
1. A primer pair for identifying or assisting in identifying polygonatum cyrtonema is characterized by comprising primers PCY1-F and PCY1-R, wherein the primers PCY1-F are (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in a sequence 2 in a sequence table;
(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;
the primer PCY1-R is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in a sequence 3 in a sequence table;
(a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3.
2. The primer pair of claim 1, for use in identification or auxiliary identification of polygonatum cyrtonema.
3. The application of claim 2, wherein the chloroplast genome of the polygonatum cyrtonema is a DNA molecule shown as a sequence 1 in a sequence table.
4. A method for identifying or assisting in identifying polygonatum cyrtonema includes the following steps:
(1) performing PCR amplification on the genomic DNA of a sample to be tested by using the primer according to claim 1;
(2) if the amplification product contains a DNA fragment of about 1500bp, the sample to be detected is or is selected as polygonatum cyrtonema; if the amplified product does not contain a DNA fragment of about 1500bp, the sample to be detected is the polygonatum cyrtonema which is not polygama.
5. The method for identifying or assisting in identifying polygonatum cyrtonema according to claim 4, wherein the genotype of a specific SNP site in the genomic DNA of a sample to be tested is CT genotype, and the sample to be tested is polygonatum cyrtonema;
the genotype of a specific SNP site in the genome DNA of a sample to be detected is a non-CT genotype, and the sample to be detected is non-polygonatum cyrtonema.
6. The method for identifying or assisting in identifying polygonatum cyrtonema according to claim 5, wherein the specific SNP site is the 153809 th nucleotide site from the 5' end in the sequence 1.
7. The method for identifying or assisting in identifying Polygonatum cyrtonema Hua according to claim 6, wherein the DNA fragment of about 1500bp is 1507 bp.
8. The method for identifying or assisting in identifying polygonatum cyrtonema according to claim 4, wherein the sample to be tested is polygonatum cyrtonema, polygonatum kingianum.
9. A kit for identifying or assisting in identifying polygonatum cyrtonema, which is characterized by comprising the primer pair according to claim 1.
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| CN114480714A (en) * | 2022-02-23 | 2022-05-13 | 合肥仟金草生物科技有限公司 | Chloroplast genome for identifying Jiuhua siberian solomonseal rhizome and application thereof in identifying Jiuhua siberian solomonseal rhizome |
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| CN109371154A (en) * | 2018-11-19 | 2019-02-22 | 浙江省林业科学研究院 | Identify characteristic sequence, primer and the method for polygonatum filipes and polygonatum cyrtonema |
| CN109762918A (en) * | 2019-01-22 | 2019-05-17 | 浙江省林业科学研究院 | Identify characteristic sequence, primer and the method for polygonatum filipes and polygonatum cyrtonema |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274245A (en) * | 2015-11-23 | 2016-01-27 | 中国中医科学院中药研究所 | Method for authenticating polygonatum cyrtonema and special primer pair thereof |
| CN109371154A (en) * | 2018-11-19 | 2019-02-22 | 浙江省林业科学研究院 | Identify characteristic sequence, primer and the method for polygonatum filipes and polygonatum cyrtonema |
| CN109762918A (en) * | 2019-01-22 | 2019-05-17 | 浙江省林业科学研究院 | Identify characteristic sequence, primer and the method for polygonatum filipes and polygonatum cyrtonema |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114480714A (en) * | 2022-02-23 | 2022-05-13 | 合肥仟金草生物科技有限公司 | Chloroplast genome for identifying Jiuhua siberian solomonseal rhizome and application thereof in identifying Jiuhua siberian solomonseal rhizome |
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