CN114438240A - High-resolution melting curve identification primer for dendrobium officinale, application and identification method - Google Patents
High-resolution melting curve identification primer for dendrobium officinale, application and identification method Download PDFInfo
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Abstract
The invention relates to a dendrobium officinale high-resolution melting curve identification primer, application and an identification method, wherein the nucleotide sequence of an upstream primer TPHML-F is shown as SEQ ID No.1, and the nucleotide sequence of a downstream primer TPHML-R is shown as SEQ ID No. 2. The identification primer can be used for identifying dendrobium officinale medicinal materials and processed products thereof, namely dendrobium officinale bailer and related species thereof. The method has the advantages of simple and convenient operation, short time consumption, low detection limit, high accuracy and good application prospect.
Description
Technical Field
The invention belongs to the technical field of molecular identification of dendrobium officinale, and particularly relates to a high-resolution melting curve identification primer for dendrobium officinale, application of the primer, and a method for identifying authenticity and adulteration of dendrobium officinale by using the primer.
Background
Dendrobe is listed as the first of the nine-big-fairy grass of China by the classic Daojia medical science (Daozang), and listed as the superior product from Shen nong Ben Cao Jing. Modern clinical researches find that polysaccharides, mannose and the like in the dendrobium have the functions of resisting hypertension and enhancing immunity, can also treat chronic pharyngitis, digestive system diseases and the like, and has very obvious effect. The dendrobium plants in the world are about 1500, all the species under the dendrobium plants are listed in appendix of International trade convention on endangered wild animals and plants, the dendrobium varieties in China only account for about 5 percent of the world, and the dendrobium officinale, the dendrobium candidum and the like with medicinal values are listed in a national list of important protected wild medicinal material species and are national grade III protected medicinal materials. In the first part of the 'Chinese pharmacopoeia' of the 2020 edition, 5 dendrobium varieties are collected, wherein the similar varieties of dendrobium jinjiangensis, dendrobium huoshanense, dendrobium chrysotoxum, dendrobium fimbriatum and congeneric plants thereof are listed under the item of 'dendrobium nobile', and the similar varieties of dendrobium officinale are listed under the item of 'dendrobium officinale'. The dendrobium officinale is a dried stem of Dendrobii officinale Kimura et Migo which belongs to the family Orchidaceae, the name is obtained because the epidermis of the dendrobium officinale is iron cyan, the dendrobium officinale has unique medicinal value by using the stem as a medicine, is a traditional famous and precious Chinese medicinal material, and has extremely high medicinal value. Due to slow propagation of the dendrobium officinale and the addition of the unregulated picking, the wild resources are scarce, and the wild resources are basically extinct and listed in the Chinese plant Red book, and become endangered rare medicinal materials; because of the similar shapes of the plants in the same genus and the similar genus of the same family, there are many confused products and counterfeit products in the market, and the processed product of the dendrobium candidum is often counterfeit by other dendrobium candidum medicinal materials. Therefore, there is a need to develop a method for identifying the authenticity and adulteration of dendrobium officinale medicinal materials and dendrobium officinale kimura et migo processed products thereof.
High resolution melting curve analysis (HRM) is an experimental conclusion based on real-time fluorescence quantitative PCR technology combined with fluorescent fuel to analyze the melting curve change of PCR products. Different PCR products contain slightly different GC contents and different base sequences, so that the Tm values of the products are different, the expressed fluorescent signals are different in intensity, and the difference among sequences can be obtained. The new gene analysis technology for forming different form melting curves by different melting temperatures of mononucleotide does not need to use a sequence specific probe, but utilizes a saturated fuel to analyze PCR reaction products, has extremely high sensitivity, can detect the difference of single base, and has the advantages of low cost, high flux, high speed, accurate result and no limitation of detection sites.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a dendrobium officinale high-resolution melting curve identification primer and a method for identifying the authenticity and adulteration of dendrobium officinale by using the primer. The method is realized by the following technical scheme:
the invention provides a group of primers for identifying a high-resolution melting curve of dendrobium officinale, wherein the primers are TPHML, and the nucleotide sequences are as follows:
the upstream primer TPHML-F: CATCCCATCCATCTGGAAATCT, as shown in SEQ ID No. 1;
the downstream primer TPHML-R: AACAATCGCAAGAAATGCAAAG, SEQ ID No. 2.
The primers are characterized in that homologous comparison is carried out on matK sequences of dendrobium officinale and congeneric species thereof in a GeneBank database by using BioEdit software, manual proofreading is assisted, stable difference SNP sites of the dendrobium officinale and the congeneric species of the dendrobium officinale are obtained through analysis, a high-resolution melting curve identification primer of the dendrobium officinale is designed on the basis of PrimerPrmier5.0 software, and whether Tm values of dendrobium officinale medicinal materials in different production places and dendrobium officinale sample processed products thereof are 75.75 +/-0.04 ℃ is verified. Therefore, the primer pair has extremely high specificity, and HRM analysis is carried out after PCR amplification is carried out on the DNA of the dendrobium officinale medicinal material and the dendrobium officinale sample of the processed product of the dendrobium officinale medicinal material by using the primer pair, only the Tm value of the dendrobium officinale medicinal material and the dendrobium officinale sample of the processed product of the dendrobium officinale sample is 75.75 +/-0.04 ℃, and the Tm value of other mixed counterfeit products is 76.53 +/-0.06 ℃.
In a second aspect of the invention, a kit for identifying dendrobium officinale is provided, and the kit comprises the primer pair.
The third aspect of the invention provides an application of the primer pair or the kit in identifying authenticity and adulteration of dendrobium officinale.
Further, the dendrobium officinale comprises dendrobium officinale medicinal materials and dendrobium officinale bailer processed products thereof.
The fourth aspect of the invention provides a method for identifying the authenticity and adulteration of dendrobium officinale by utilizing a high-resolution melting curve analysis technology, which comprises the following steps: extracting DNA of a sample to be detected; mixing the DNA of a sample to be detected as template DNA with the primer pair or the primer pair in the kit, and then carrying out PCR amplification; and (3) entering a high-resolution melting curve analysis program after the PCR is finished, and if the Tm value of a sample to be detected is 75.75 +/-0.04 ℃, determining that the sample to be detected is the dendrobium officinale, wherein the sample to be detected comprises the dendrobium officinale medicinal material and the processed product of the dendrobium officinale.
Further, the method for extracting the DNA of the sample to be tested is not particularly limited in the present invention, and extraction by the CTAB method is preferable.
Further, in the present invention, the method for extracting DNA from the sample to be tested is preferably, the sample to be tested is 20-100mg, ground by a high throughput tissue grinder, and then extracted by a two-step CTAB method for total DNA, diluted to obtain template DNA.
Further, the PCR amplification reaction program comprises: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 40s, and extension at 72 ℃ for 40s for 40 cycles; the extension was carried out for a further 7min at 72 ℃.
Further, in the present invention, the PCR amplification reaction system comprises, in 20 μ L: 2 × High Resolution Messing Master Mix 10 μ L, MgCl21.6. mu.L (2.5mmol/L), 0.4. mu.L each of primers (10. mu. mol/L), 1. mu.L of template DNA, ddH2O 6.6μL。
Further, in the present invention, the concentration of the template DNA is 1 ng. mu.L-1～100 ng·μL-1。
After the PCR amplification, the method also comprises the step of performing HRM analysis on the PCR amplification product; the HRM analysis program includes: at 0.02℃·s-1The speed is increased from 70 ℃ to 90 ℃, and fluorescence is collected for 25 times at 1 ℃ per liter. If the Tm value of the sample to be detected is 75.75 +/-0.04 ℃, the sample is the dendrobium officinale, otherwise, the sample is the dendrobium officinale pseudo-mixture.
The invention has the following beneficial effects:
1) the high-resolution melting curve identification primer TPHML-F/R can be used for identifying the authenticity and adulteration of the dendrobium officinale and the dendrobium officinale processed products thereof from the dendrobium officinale and the dendrobium officinale species, and has the advantages of extremely high specificity, simple method, low cost, high efficiency, accurate result and no restriction of detection sites;
2) when the DNA concentration of the adulterant in the dendrobium officinale and processed products thereof, namely dendrobium officinale bucket adulterant mixed samples is more than or equal to 1 percent, the method disclosed by the invention can accurately detect the adulterant, has low detection limit and has good application prospect;
3) the method is not influenced by the concentration of the sample DNA, has good stability, and has the concentration range of 1-100 ng.mu.L when the concentration of the sample DNA is-1In time, the mixed counterfeit products of dendrobium officinale and common dendrobium and the mixed counterfeit products of dendrobium officinale and other dendrobium medicinal materials can be accurately identified.
Drawings
FIG. 1 shows the effect of different primers and template DNA PCR amplification on Tm values of Dendrobium officinale and mixed counterfeit products;
FIG. 2 is a diagram of a standard HRM melting curve of Dendrobium officinale;
FIG. 3 is a HRM differential curve diagram of Dendrobium officinale and mixed counterfeit products with different DNA concentrations; FIG. 3(A) shows that the DNA concentration was 1 ng. mu.L-1HRM differential curve chart of dendrobium officinale and mixed counterfeit products; FIG. 3(B) shows the DNA concentration of 5 ng. mu.L-1HRM differential curve chart of dendrobium officinale and mixed counterfeit products; FIG. 3(C) shows that the DNA concentration was 25 ng. mu.L-1HRM differential curve diagram of dendrobium officinale and mixed counterfeit products; FIG. 3(D) shows the DNA concentration of 50 ng. mu.L-1HRM differential curve chart of dendrobium officinale and mixed counterfeit products; FIG. 3(E) shows the DNA concentration of 100 ng. mu.L-1HRM differential curve chart of dendrobium officinale and mixed counterfeit products;
FIG. 4 is a graph showing how the DNA of the dendrobium officinale adulterant and the DNA of the dendrobium officinale sample are mixed together as a template in different concentration ratios; FIG. 4(A) is a normalized graph of the mixture of DNA of Dendrobium officinale pseudo-mixture and DNA of Dendrobium officinale sample as template; FIG. 4(B) is a difference curve diagram of the mixture of DNA of the dendrobium officinale pseudo-mixture and DNA of the dendrobium officinale sample as a template in different concentration ratios;
FIG. 5 is a graph showing the identification of Dendrobium officinale and counterfeit drugs; FIG. 5(A) is a melting curve diagram for identifying Dendrobium officinale and counterfeit mixture; FIG. 5(B) is a normalized graph for identifying Dendrobium officinale and counterfeit mixture; FIG. 5(C) is a differential curve diagram for identifying Dendrobium officinale and counterfeit mixture.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings in order to better understand the technical solution.
According to the invention, a BioEdit software is used for carrying out homologous comparison on matK sequences of dendrobium officinale and congeneric species thereof in a GeneBank database, manual proofreading is assisted, stable difference SNP sites of the dendrobium officinale and the congeneric species of the dendrobium officinale are obtained through analysis, 2 pairs of high-resolution melting curve identification primers of the dendrobium officinale are designed based on a PrimerPrmier5.0 software, and the sequences of the primers and the annealing temperature are shown in a table 1.
TABLE 1 primer sequences and annealing temperatures
In order to investigate the difference significance of 2 pairs of primers in table 1 in identifying the mixed counterfeit products of dendrobium officinale and the closely related dendrobium species, the invention performs PCR amplification and HRM analysis on 2 pairs of primers in table 1, and analyzes the peak shape and Tm value of the primers, and the result shows that after the primers TPHML-1F/R are amplified, the Tm value of a dendrobium officinale sample is 74.99 +/-0.11 ℃, and the Tm value of a mixed counterfeit product sample is 75.87 +/-0.12 ℃; after the primer TPHML-2F/R is amplified, the Tm value of the dendrobium officinale sample is 75.75 +/-0.04 ℃, the Tm value of the mixed counterfeit sample is 76.53 +/-0.06 ℃, the Tm value of the mixed counterfeit sample is more concentrated, and the difference with the Tm value of the dendrobium officinale is more obvious, as shown in figure 1. Therefore, the preferable primer TPHML-2F/R is used as the group of dendrobium officinale high-resolution dissolution curve identification primers.
In order to construct a standard high-resolution melting curve model of the dendrobium officinale, DNA is extracted from 30 parts of dendrobium officinale medicinal materials in different producing areas and 12 parts of dendrobium officinale maple bucket samples, HRM analysis is carried out by using Roche 480Ver.1.2 software, and the standard high-resolution melting curve model of the dendrobium officinale is established according to the extracted DNA, wherein the sample with the fluorescence collection value lower than 60% of the maximum value is not listed in an analysis range. The results show that the peaks of the obtained dendrobium officinale standard high-resolution melting curve model are consistent and are all single peaks, the Tm values of the dendrobium officinale standard high-resolution melting curve model are 75.75 ℃, and the standard deviation is 0.04, which is shown in figure 2.
In order to investigate the stability of the method, the DNA templates of dendrobium officinale and common dendrobium blended counterfeit products are respectively diluted to be 100 ng.mu.L-1、50ng·μL-1、 25ng·μL-1、5ng·μL-1、1ng·μL-1And performing HRM differential curve analysis after PCR amplification. The results show that the concentration of the DNA template is in the range of 1 to 100 ng. mu.L-1In the process, the dendrobium officinale and common dendrobium mixed counterfeit products can be accurately typed, the curve shape difference is large, and the sample DNA concentration is more stable when being low, as shown in figure 3. The method for identifying the dendrobium officinale by using the high-resolution dissolution curve analysis technology is good in stability.
In order to investigate the detection capability of the method of the invention on the counterfeit in the adulterated sample, the DNA of the common dendrobium adulterated sample is mixed with the DNA of the dendrobium officinale sample according to the concentration proportion of 0%, 1%, 5%, 25%, 50%, 99% and 100% to be used as a DNA template, and the genotyping analysis is carried out after the amplification of a group of dendrobium officinale high-resolution melting curve identification primers. The result shows that when the concentration content of the DNA of the dendrobium officinale pseudo-mixture in the mixed sample is only 1%, the mixed sample can be distinguished from the dendrobium officinale genuine product through genotyping, and the difference with the dendrobium officinale is larger as the concentration content of the DNA of the dendrobium officinale pseudo-mixture is higher; when the concentration of the DNA of the counterfeit in the adulteration mixed sample is more than or equal to 1 percent, the method can accurately detect the counterfeit, which is shown in figure 4. The method for identifying the dendrobium officinale by using the high-resolution melting curve analysis technology has a good detection effect on the dendrobium officinale adulterant.
Example 1: a method for identifying Dendrobium officinale by using high-resolution melting curve technology comprises the following steps: the dendrobium officinale adopted in the embodiment is from XianHu No.2 of the organic national medicine health-preserving base for producing longevity immortal grains.
The 7 common Dendrobium adulterants adopted in this example are Dendrobium nobile (Dendrobium nobilendl.), Dendrobium huoshanense (Dendrobium huoshanense ec.z.tang et s.j.cheng), Dendrobium chrysotoxum (Dendrobium chrysotoxum lindl.), Dendrobium fimbriatum (Dendrobium fimbriatum Hook.), Dendrobium guangdongensis (Dendrobium wilsonii Rolfe.), Dendrobium candidum (Dendrobium gratissimum rchb.f.), Dendrobium chrysanthum (Dendrobium grandiflorum linum), Dendrobium chrysanthum (Dendrobium chrysogenum linum), Dendrobium candidum (Dendrobium chrysogenum linum), and Dendrobium devonianum paxt).
Experimental methods
(1) Extracting DNA of a sample to be detected: taking 20-100mg of dendrobium officinale original plants and 7 common dendrobium mixed counterfeit products, grinding by using a high-throughput tissue grinder, extracting total DNA of a sample by using a two-step CABT method, detecting the concentration of the DNA by using a NanoDropTM one type ultramicro ultraviolet-visible spectrophotometer of the U.S. Thermo company, and then diluting to 100 ng. mu.L-1、50 ng·μL-1、25ng·μL-1、5ng·μL-1、1ng·μL-1Obtaining template DNA, and placing in a refrigerator at 4 ℃ for later use.
(2) Designing and synthesizing a primer: performing homologous comparison on matK sequences of dendrobium officinale and congeneric and homologous species thereof in a GeneBank database by using BioEdit software, performing manual proofreading, analyzing to obtain stable difference SNP sites of the dendrobium officinale and the allied species, designing a high-resolution melting curve identification primer of the dendrobium officinale based on Primer Prmier5.0 software, and performing upstream primer TPHML-F: 5'-CATCCCATCCATCTGGAAATCT-3' and the downstream primer TPHML-R: 5'-AACAATCGCAAGAAATGCAAAG-3', synthesizing a primer.
(3) PCR amplification of primer pair (TPHML-F/R): after mixing the template DNA sample with the primer pair (TPHML-F/R), PCR amplification and high resolution melting curve analysis were performed on a Light Cycler 480 type fluorescent quantitative PCR instrument.
PCR amplification reaction system (total volume 20. mu.L): 2 × High Resolution Messing Master Mix 10 μ L, MgCl2(2.5mmol/L) 1.6. mu.L, forward primer (10)μ mol/L)0.4 μ L, downstream primer (10 μmol/L)0.4 μ L, template DNA 1 μ L, ddH2O 20μL。
The PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 40s, and extension at 72 ℃ for 40s for 40 cycles; the extension was carried out for a further 7min at 72 ℃.
(4) High resolution melting curve analysis: after the PCR amplification of the primer pair (TPHML-F/R) is finished, HRM analysis is carried out by using Roche 480Ver.1.2 software, wherein the analysis program comprises the following steps: the temperature is increased from 70 ℃ to 90 ℃ at the speed of 0.02 ℃/s, and the fluorescence is collected for 25 times at the temperature of 1 ℃ per liter.
The experimental result is shown in fig. 5A, the melting curves of the dendrobium officinale and the 7 counterfeit mixture samples are all single peaks, but the peak positions are obviously different, the Tm value of the dendrobium officinale is 75.75 +/-0.04 ℃, and the Tm value of the counterfeit mixture sample is 76.53 +/-0.06 ℃.
In order to eliminate the influence of the concentration of the DNA of the sample on the identification result, the fusion curve is subjected to normalization processing and differential analysis, the result shows that the difference between the normalization curve and the differential curve shape of the dendrobium officinale and the mixed counterfeit product is obvious, and the fusion curve, the normalization curve and the differential curve can accurately distinguish the dendrobium officinale and the common dendrobium mixed counterfeit product, as shown in fig. 5B and 5C.
Claims (10)
1. A specific molecular marker TPHML for identifying dendrobium officinale is characterized in that the molecular marker comprises an upstream primer TPHML-F and a downstream primer TPHML-R, wherein the nucleotide sequence of the upstream primer TPHML-F is shown as SEQ ID NO. 1; the nucleotide sequence of the downstream primer TPHML-R is shown as SEQ ID NO. 2.
2. The kit for identifying the dendrobium officinale is characterized by comprising a molecular marker TPHML, wherein the molecular marker comprises an upstream primer TPHML-F and a downstream primer TPHML-R, and the nucleotide sequence of the upstream primer TPHML-F is shown as SEQ ID No. 1; the nucleotide sequence of the downstream primer TPHML-R is shown as SEQ ID NO. 2.
3. The primer pair of claim 1 or the kit of claim 2 is used for identifying authenticity and adulteration of dendrobium officinale.
4. The use of claim 3, wherein the Dendrobium officinale comprises Dendrobium officinale and its processed product Dendrobium officinale.
5. A method for identifying dendrobium officinale by using a high-resolution melting curve analysis technology is characterized by comprising the following steps:
1) extracting DNA of a sample to be detected;
2) mixing DNA of a sample to be detected as template DNA with a molecular marker TPHML, and then carrying out PCR amplification;
3) high-resolution melting curve analysis: and (4) after the PCR amplification is finished, entering a high-resolution melting curve analysis program, and if the Tm value of the sample to be detected is 75.75 +/-0.04 ℃, determining the sample is the dendrobium officinale.
6. The method for identifying Dendrobium officinale Kimura et Migo by using high resolution melting curve analysis technology as claimed in claim 5, wherein in step 1), the amount of the sample to be detected is 20-100mg, after grinding with a high throughput tissue grinder, the total DNA of the sample is extracted by using a two-step CTAB method, and the template DNA is obtained by dilution.
7. The method of claim 5, wherein the PCR amplification reaction process in step 2) comprises: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 40s, and extension at 72 ℃ for 40s for 40 cycles; the extension was carried out for a further 7min at 72 ℃.
8. The method for identifying Dendrobium officinale Kimura et Migo by using high resolution melting curve analysis technology as claimed in claim 5, wherein the PCR amplification reaction system in step 2) is in 20 μ L, comprising: 2 × High Resolution Messing Master Mix 10 μ L, MgCl21.6. mu.L (2.5mmol/L), 0.4. mu.L each of primers (10. mu. mol/L), 1. mu.L of template DNA, ddH2O 6.6μL。
9. The method of claim 5, wherein the step 3) of analyzing the high resolution melting curve comprises: at 0.02 ℃ s-1The speed is increased from 70 ℃ to 90 ℃, and fluorescence is collected for 25 times when the temperature per liter is increased by 1 ℃.
10. The method of claim 8, wherein the template DNA is at a concentration of 1 ng. mu.L-1 ～100 ng·μL-1。
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| CN101810140A (en) * | 2009-09-03 | 2010-08-25 | 四川万安石斛产业开发有限公司 | Method for propagating dendrobium candidum test-tube plantlets |
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