CN107630068B - Dendrobium huoshanense specific molecular marker and detection method thereof - Google Patents
Dendrobium huoshanense specific molecular marker and detection method thereof Download PDFInfo
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Abstract
The invention discloses a dendrobium huoshanense specific molecular marker and a detection method thereof. The invention provides a method for identifying or assisting in identifying dendrobium huoshanense, which comprises the following steps: extracting the genome DNA of a sample to be detected, taking the genome DNA of the sample to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer HSSH-1.F and a primer HSSH-1.R, wherein if an amplification product contains a DNA fragment of 200bp-300bp, the sample to be detected is dendrobium huoshanense or is a candidate, and if the amplification product does not contain the DNA fragment of 200bp-300bp, the sample to be detected is dendrobium huoshanense or is a candidate; the primer HSSH-1.F is shown as a sequence 2; the HSSH-1.R is shown as a sequence 3. The method established by the invention has extremely high specificity and can be used as a specific molecular marker of dendrobium huoshanense.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine molecular identification, and particularly relates to a dendrobium huoshanense specific molecular marker and a detection method thereof.
Background
The Dendrobium huoshanense is derived from fresh or dried stem of Dendrobium huoshanense C.Z.Tang et S.J.Cheng. The dendrobium huoshanense is the top product of dendrobium medicinal materials, has good medicinal and health-care values, large market demand and high price, and the price of a fresh product exceeds 30 yuan per gram. The phenomenon that the dendrobium plant mixed beads of other species of the dendrobium plant are used as dendrobium huoshanense is frequently found in the market. Dendrobium officinale D.officinale Kimura et Migo, Dendrobium moniliforme D.moniliforme (L.), sweet, D.citrifolia D.tosarene Makino, Dendrobium bicolor D.heroglossum Reichb, Dendrobium officinale D.loddigesii Rolfe, Dendrobium loddigesii Rolfe, Dendrobium nobile D.hancocockii Rolfe, Dendrobium Cantoneum D.wilsonii Rolfe, Dendrobium loddigesii D.lohennse T.Tang et F.T.Wang, Dendrobium Henan D.hennense J.L.Lu et L.X.Gao, etc., which are similar in form and chemical composition, especially after the processed product is made into a sweet gum bucket and a dry strip, the original plant morphological characteristics are destroyed, the traditional morphological and chemical identification is difficult to distinguish, and the dendrobium loddigesii is usually filled in the commercially available decoction pieces.
At present, people mainly distinguish dendrobium huoshanense and mixed counterfeit products thereof by a traditional character identification method, and the identification accuracy is insufficient and the subjectivity is strong. In addition, there are reports of identifying dendrobium huoshanense by using ISSR-PCR amplification map or ITS sequence-based site-specific PCR technology. However, the latest molecular systematic theory shows that dendrobium huoshanense belongs to a dendrobium huoshanense complex, has no difference on nuclear genes which are irrelevant to properties such as ITS and the like, and cannot be distinguished through markers on ITS sequences.
Disclosure of Invention
The invention aims to provide a dendrobium huoshanense specific molecular marker and a detection method thereof.
The invention provides a method for identifying or assisting in identifying dendrobium huoshanense, which comprises the following steps: extracting the genome DNA of a sample to be detected, taking the genome DNA of the sample to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer HSSH-1.F and a primer HSSH-1.R, wherein if an amplification product contains a DNA fragment of 200bp-300bp, the sample to be detected is dendrobium huoshanense or is a candidate, and if the amplification product does not contain the DNA fragment of 200bp-300bp, the sample to be detected is dendrobium huoshanense or is a candidate.
The DNA fragment of 200bp-300bp can be 213 bp.
The DNA fragment of 200bp-300bp can be specifically shown as a sequence 4.
The invention also provides a method for identifying or assisting in identifying dendrobium huoshanense, which comprises the following steps: detecting the genotype of a specific SNP locus in the genome DNA of a sample to be detected, wherein if the genotype of the specific SNP locus is GG genotype, the sample to be detected is dendrobium huoshanense or a candidate thereof, and if the genotype of the specific SNP locus is non-GG genotype, the sample to be detected is dendrobium huoshanense or a candidate thereof;
the method for detecting the genotype of the specific SNP locus in the genome DNA of the sample to be detected comprises the following steps: extracting the genome DNA of a sample to be detected, carrying out PCR amplification by using the genome DNA as a template and a primer pair consisting of a primer HSSH-1.F and a primer HSSH-1.R, and then sequencing the PCR amplification product.
The non-GG genotype may specifically be the TT genotype.
The invention also provides a method for detecting the specific SNP locus in the genome DNA of the dendrobium plant, which comprises the following steps: extracting the genome DNA of a sample to be detected, carrying out PCR amplification by using the genome DNA as a template and a primer pair consisting of a primer HSSH-1.F and a primer HSSH-1.R, and then sequencing the PCR amplification product.
The invention also provides a method for detecting the specific SNP locus in the genome DNA of the dendrobium plant, which comprises the following steps: extracting the genome DNA of a sample to be detected, taking the genome DNA of the sample to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer HSSH-1.F and a primer HSSH-1.R, wherein if an amplification product contains a DNA fragment of 200bp-300bp, the genotype of a specific SNP site in the genome DNA of the sample to be detected is GG, and if the amplification product does not contain the DNA fragment of 200bp-300bp, the genotype of the specific SNP site in the genome DNA of the sample to be detected is non-GG.
The DNA fragment of 200bp-300bp can be 213 bp.
The DNA fragment of 200bp-300bp can be specifically shown as a sequence 4.
The non-GG may specifically be TT.
Any one of the specific SNP sites is 190 th nucleotide from the 5' end of the sequence 1 in the sequence table in the genome of the dendrobe plant.
Any one of the primers HSSH-1.F is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a2) and (b) a DNA molecule which is obtained by substituting the sequence 2 by one or more nucleotides and has the same function as the sequence 2.
Any one of the primers HSSH-1.R is (a3) or (a 4):
(a3) a single-stranded DNA molecule shown in sequence 3 of the sequence table;
(a4) and (b) a DNA molecule which is obtained by substituting the sequence 3 by one or more nucleotides and has the same function as the sequence 3.
The invention also protects a specific DNA molecule which is shown as a sequence 1 in a sequence table.
The invention also protects the application of the specific DNA molecule in identification or assisted identification of dendrobium huoshanense.
When identifying or assisting in identifying dendrobium huoshanense, if the 190 th nucleotide at the 5' tail end of the specific DNA molecule in the sample to be detected is G, the sample to be detected is or is candidate to be dendrobium huoshanense; if the 190 th nucleotide at the 5' end of the specific DNA molecule in the sample to be detected is T, the sample to be detected is or is selected as non-dendrobium huoshanense.
The invention also protects a specific primer pair, which consists of a primer HSSH-1.F and a primer HSSH-1. R;
the primer HSSH-1.F is (a1) or (a 2):
(a1) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a2) DNA molecule which is obtained by substituting one or more nucleotides in the sequence 2 and has the same function as the sequence 2;
the primer HSSH-1.R is (a3) or (a4) as follows:
(a3) a single-stranded DNA molecule shown in sequence 3 of the sequence table;
(a4) and (b) a DNA molecule which is obtained by substituting the sequence 3 by one or more nucleotides and has the same function as the sequence 3.
The invention also protects the application of the specific primer pair in identification or assisted identification of dendrobium huoshanense.
The invention also protects a kit containing the specific primer pair; the application of the kit is identification or auxiliary identification of dendrobium huoshanense.
The reaction system for any one of the above PCR amplifications may specifically be: 2.5 mu L of 10 XTaq buffer solution, 1 mu L of 2.5mmol/L dNTPs, 0.25 mu L of primer HSSH-1.F, 0.25 mu L of primer HSSH-1.R, 1U of Taq DNA polymerase, 1-100ng of DNA template and sterile distilled water to make up to 25 mu L.
The primer HSSH-1.F and the primer HSSH-1.R are both added into a PCR reaction system in the form of primer solution, and the initial concentration of the primer HSSH-1.F and the initial concentration of the primer HSSH-1.R in the primer solution are both 10 mu M.
The reaction procedure of any one of the above PCR amplifications may specifically be: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 5 min.
Any one of the above samples to be tested may specifically be any one of the following: dendrobium huoshanense, dendrobium officinale, dendrobium moniliforme, dendrobium nobile, dendrobium heyneanum, dendrobium chrysotoxum, dendrobium devonianum, dendrobium loddigesii, dendrobium candidum, dendrobium devonianum, dendrobium nobile, dendrobium globosum, dendrobium chrysanthum robustum, dendrobium nobile lindl, dendrobium fimbriatum, dendrobium nobile and dendrobium nobile.
The invention carries out sequencing and sequence analysis on 68 dendrobium samples containing all species of dendrobium huoshanense complex by sequencing and sequence analysis technology, finds out an SNP molecular marker only existing in dendrobium huoshanense, and establishes a specific detection method of the SNP marker.
Drawings
FIG. 1 shows the specific PCR electrophoresis detection results of some tested dendrobe samples. Wherein M is DL2000DNA molecular weight standard (the sizes of bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom respectively); lanes 1-4 are dendrobium huoshanense samples, 1: dendrobium huoshanense (numbered as 1 in table 2); 2: dendrobium huoshanense (numbered 6 in table 2); 3: dendrobium huoshanense (numbered as 12 in table 2); 4: dendrobium huoshanense (numbered 28 in table 2); lanes 5-23 are other dendrobe samples, 5: dendrobium chrysotoxum (numbered 61 in table 2); 6: dendrobium devonianum (numbered 56 in Table 2); 7: dendrobium nobile lindl (number 66 in table 2); 8: dendrobium aphyllum (numbered 72 in table 2); 9: dendrobium candidum (numbered as 77 in table 2); 10: dendrobium chrysanthum (numbered 81 in table 2); 11: dendrobium nobile (numbered 83 in Table 2); 12: dendrobium fimbriatum (numbered 85 in table 2); 13: dendrobium huoshanense (numbered 89 in Table 2); 14: dendrobium candidum (numbered 91 in table 2); 15: dendrobium loddigesii (numbered 94 in table 2); 16: dendrobium huoshanense (numbered as 99 in table 2); 17: dendrobium loddigesii Rolfe (numbered 97 in Table 2); 18: dendrobium nobile lindl (numbered 98 in table 2); 19: dendrobium mengyenium (numbered 100 in table 2); 20: dendrobium primula (numbered 102 in Table 2); 21: dendrobium officinale (numbered as 41 in table 2); 22: dendrobium moniliforme (number 51 in table 2); 23: dendrobium huinan (numbered 53 in table 2); n is blank control (using equal volume of ddH)2O as a template).
FIG. 2 shows the sensitivity experiment of Dendrobium huoshanense primer pair. Wherein M is DL2000DNA molecular weight standard (the band sizes are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom respectively).
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The dendrobe plants in the following examples are all incorporated by reference: xu S, Li D, Li J, et al. evaluation of the DNA barcodes in Dendrobium (Orchidaceae) from mainland Asia [ J ]. Plos One, 2015, 10 (1): e0115168-e0115168. published; the public is available from the institute of traditional Chinese medicine of the Chinese academy of sciences.
10 × Taq buffer, Taq DNA polymerase: takara, catalog number R001A.
Example 1 Dendrobium huoshanense specific SNP site
1. 68 dendrobium samples containing all species of dendrobium huoshanense complex are sequenced and subjected to sequence analysis, and a SNP molecular marker only existing in dendrobium huoshanense is found on a genome sequence.
The SNP site and the nucleotides near the SNP site are shown as a sequence 1 in a sequence table, wherein the 190 th nucleotide is the SNP site and is G/T polymorphism.
2. Primers were designed for the SNP sites of step 1, as shown in Table 1.
TABLE 1 primer information
3. The establishment of the method for identifying dendrobium huoshanense comprises the following steps:
(1) extracting the genome DNA of a sample to be detected;
(2) and (3) performing PCR amplification by using the genomic DNA obtained in the step (1) as a template and adopting the primer HSSH-1.F and the primer HSSH-1.R prepared in the step (2) to obtain an amplification product.
PCR amplification reaction (25. mu.L): 2.5 μ L of 10 XTaq buffer, 1 μ L of 2.5mmol/L dNTPs, 0.25 μ L of primer HSSH-1.F, 0.25 μ L of primer HSSH-1.R, 1U of Taq DNA polymerase, about 20ng of DNA template, and sterile distilled water to make up to 25 μ L. The primer HSSH-1.F and the primer HSSH-1.R are both added into a PCR reaction system in the form of primer solution, and the initial concentration of the primer HSSH-1.F and the primer HSSH-1.R in the primer solution is 10 mu M.
PCR amplification reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 5 min.
(3) And (3) carrying out 1.5% 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 200bp-300bp, the sample to be detected is dendrobium huoshanense or is a candidate for dendrobium huoshanense; if the amplified product does not contain the DNA fragment of 200bp-300bp, the sample to be detected is or is selected as non-dendrobium huoshanense.
Example 2 effective verification of method for identifying dendrobium huoshanense based on specific PCR
A sample to be detected: the 103 dendrobe materials from known species from different origins are shown in table 2. 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.
The detection method established in step 3 of embodiment 1 is adopted to detect the sample to be detected. The electrophoresis results are shown in Table 2 and FIG. 1. The results of the specific PCR detection by the method of example 1 are consistent with the actual conditions, and the detection accuracy is up to 100%. FIG. 1 shows the result of electrophoresis of PCR amplification products of a part of samples.
The 213bp target bands obtained from each dendrobium huoshanense sample in table 2 were sequenced, and the sequencing results are all shown in sequence 4 (the 190 th base is G).
Table 1103 dendrobe samples from known species in different producing areas and specific PCR detection results thereof
Example 3 sensitivity
The sample to be tested is: randomly selected 2 out of 38 dendrobium huoshanense samples in table 2 (numbered 12 and 28 in table 2), randomly selected 1 out of 4 dendrobium aphyllum samples in table 2 (numbered 72 in table 2), and randomly selected 1 out of 2 dendrobium nobile samples in table 2 (numbered 83 in table 2).
1. And extracting the genome DNA of the sample to be detected.
2. By ddH2And O, diluting the DNA solution obtained in the step 1 to obtain each dilution.
3. The dilution obtained in step 2 was used as a template, and PCR amplification was performed using the primer set prepared in example 1.
PCR amplification reaction (25. mu.L): 2.5 μ L of 10 XTaq buffer, 1 μ L of 2.5mmol/L dNTPs, 0.25 μ L of primer HSSH-1.F, 0.25 μ L of primer HSSH-1.R, 1U of Taq DNA polymerase, about 20ng of DNA template, and sterile distilled water to make up to 25 μ L. The primer HSSH-1.F and the primer HSSH-1.R are both added into a PCR reaction system in the form of primer solution, and the initial concentration of the primer HSSH-1.F and the primer HSSH-1.R in the primer solution is 10 mu M.
PCR amplification reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 5 min.
Due to different dilution degrees of the adopted diluents, different reaction systems are formed as follows:
in the reaction system 1, the initial content of the genomic DNA of the sample to be detected is 100 ng;
in the reaction system 2, the initial content of the genome DNA of the sample to be detected is 33 ng;
in the reaction system 3, the initial content of the genomic DNA of the sample to be detected is 11 ng;
in the reaction system 4, the initial content of the genomic DNA of the sample to be detected is 3 ng;
in the reaction system 5, the initial content of the genomic DNA of the sample to be tested was 1 ng.
4. The amplification product of step 3 was subjected to 1.5% agarose gel electrophoresis.
The results are shown in FIG. 2. The result shows that when the content of the DNA template is 1ng-100ng, only dendrobium huoshanense can amplify a specific target band, the band brightness is weakened along with the reduction of the concentration of the DNA template, the detection limit reaches 1ng, and when the content of the template is 3ng-100ng, the result is more accurate.
<110> institute of traditional Chinese medicine of Chinese academy of traditional Chinese medicine
<120> dendrobium huoshanense specific molecular marker and detection method thereof
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Claims (10)
1. A method for identifying or assisting in identifying dendrobium huoshanense comprises the following steps: extracting genome DNA of a sample to be detected, taking the genome DNA of the sample to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer HSSH-1.F and a primer HSSH-1.R, wherein if an amplification product contains a DNA fragment of 200bp-300bp, the sample to be detected is or is a candidate of dendrobium huoshanense, and if the amplification product does not contain the DNA fragment of 200bp-300bp, the sample to be detected is or is a candidate of non-dendrobium huoshanense;
the primer HSSH-1.F is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
the primer HSSH-1.R is a single-stranded DNA molecule shown in a sequence 3 of a sequence table.
2. A method for identifying or assisting in identifying dendrobium huoshanense comprises the following steps: detecting the genotype of a specific SNP locus in the genome DNA of a sample to be detected, wherein if the genotype of the specific SNP locus is GG genotype, the sample to be detected is or is selected as dendrobium huoshanense, and if the genotype of the specific SNP locus is non-GG genotype, the sample to be detected is or is selected as non-dendrobium huoshanense;
the specific SNP site is 190 th nucleotide from 5' end of sequence 1 in a sequence table in genome of dendrobe.
3. The method of claim 2, wherein: the method for detecting the genotype of the specific SNP locus in the genome DNA of the sample to be detected comprises the following steps: extracting genome DNA of a sample to be detected, performing PCR amplification by using the genome DNA as a template and a primer pair consisting of a primer HSSH-1.F and a primer HSSH-1.R, and sequencing a PCR amplification product;
the primer HSSH-1.F is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
the primer HSSH-1.R is a single-stranded DNA molecule shown in a column 3.
4. A method for detecting specific SNP sites in genome DNA of dendrobe plants comprises the following steps: extracting genome DNA of a sample to be detected, performing PCR amplification by using the genome DNA as a template and a primer pair consisting of a primer HSSH-1.F and a primer HSSH-1.R, and sequencing a PCR amplification product;
the primer HSSH-1.F is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
the primer HSSH-1.R is a single-stranded DNA molecule shown in a sequence 3 of a sequence table;
the specific SNP site is 190 th nucleotide from 5' end of sequence 1 in a sequence table in genome of dendrobe.
5. A method for detecting specific SNP sites in genome DNA of dendrobe plants comprises the following steps: extracting the genome DNA of a sample to be detected, taking the genome DNA of the sample to be detected as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer HSSH-1.F and a primer HSSH-1.R, wherein if an amplification product contains a DNA fragment of 200bp-300bp, the genotype of a specific SNP site in the genome DNA of the sample to be detected is GG, and if the amplification product does not contain the DNA fragment of 200bp-300bp, the genotype of the specific SNP site in the genome DNA of the sample to be detected is non-GG;
the primer HSSH-1.F is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
the primer HSSH-1.R is a single-stranded DNA molecule shown in a sequence 3 of a sequence table;
the specific SNP site is 190 th nucleotide from 5' end of sequence 1 in a sequence table in genome of dendrobe.
6. A specific DNA molecule is shown as sequence 1 in a sequence table.
7. The use of the specific DNA molecule of claim 6 for identification or assisted identification of Dendrobium huoshanense.
8. The specific primer pair consists of a primer HSSH-1.F and a primer HSSH-1. R;
the primer HSSH-1.F is a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
the primer HSSH-1.R is a single-stranded DNA molecule shown in a sequence 3 of a sequence table.
9. The application of the specific primer pair of claim 8 in identification or assisted identification of dendrobium huoshanense.
10. A kit comprising a specific primer pair according to claim 8; the application of the kit is identification or auxiliary identification of dendrobium huoshanense.
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| Title |
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| Molecular Authentication of Dendrobium Species;Chu-Hui Chiang等;《J. AMER. SOC. HORT. SCI》;20121231;第137卷(第6期);第438-444页 * |
| 霍山石斛的分子鉴定及其遗传多样性评价;张潇潇;《中国优秀硕士学位论文全文数据库农业科技辑》;20130315(第3期);D047-258第1-70页 * |
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