CN120210404B - MNP (MNP) marker locus, primer group, kit and identification method for identifying traditional Chinese medicinal materials including cynanchum atratum, cynomorium sonii and cynanchum paniculatum - Google Patents
MNP (MNP) marker locus, primer group, kit and identification method for identifying traditional Chinese medicinal materials including cynanchum atratum, cynomorium sonii and cynanchum paniculatumInfo
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Abstract
The invention belongs to the technical field of molecular identification, and particularly relates to MNP (MNP) marking sites, primer groups, a kit and an identification method for identifying cynanchum atratum, cynanchum atratum and cynanchum paniculatum. The MNP marking site comprises MNP-1-MNP-20 in a genome region with a plurality of nucleotide polymorphisms in cynanchum atratum, cynanchum atratum and cynanchum paniculatum. The MNP provided by the invention has the advantages of multiple number of MNP marking sites, high polymorphism and strong variety identification capability, and meets the requirements of distinguishing similar but actually different traditional Chinese medicinal materials (cynanchum atratum, cynomorium songaricum and cynanchum paniculatum). The primer group provided by the invention can be used for obtaining DNA fingerprint data of the sample to be detected, and then the variety identification conclusion is obtained by comparing the DNA fingerprint data among the samples to be detected. The detection method can compare hundreds to thousands of samples to be detected at one time, can rapidly obtain variety identification conclusion, and can remarkably improve accuracy and efficiency of identification of traditional Chinese medicinal materials including cynanchum atratum, cynomorium songaricum and cynanchum paniculatum.
Description
Technical Field
The invention belongs to the technical field of molecular identification, and particularly relates to MNP (MNP) marking sites, primer groups, a kit and an identification method for identifying cynanchum atratum, cynanchum atratum and cynanchum paniculatum.
Background
The cynanchum atratum, the cynomorium venetum and the cynanchum paniculatum are derived from the same family and genus plants, all belong to the genus goose down of the family Asclepiadaceae, have very similar appearance characters, and are easy to be confused in the purchasing, distributing and application processes. The rhizoma Cynanchi Stauntonii has medicinal value for warming and resolving cold phlegm, and is derived from root and rhizome of Cynanchum Wilfordii leaf and Cynanchum Wilfordii leaf of perennial herb of Asclepiadaceae. The radix Cynanchi Atrati has medicinal value of clearing and removing deficiency heat, and is root and rhizome of radix Cynanchi Atrati or radix Cynanchi Atrati of perennial herb of Asclepiadaceae. The Laogui has medicinal value of relieving cough and asthma, resisting inflammation and inhibiting bacteria, and is derived from dry aerial parts of perennial herb of Asclepiadaceae, cynanchum otophyllum. The radix Cynanchi Paniculati has analgesic, antibacterial, and dampness eliminating medicinal value, and is derived from dried root or rhizome of radix Cynanchi Paniculati of Asclepiadaceae.
The morphological characters of cynanchum atratum, cynanchum atratum and cynanchum paniculatum are similar, but the medicinal components are different, and the medicinal effects are not completely the same. The cynanchum atratum contains triterpenoid saponin, flavonoid glycoside and other components, is a qi-flowing-reducing phlegm-resolving medicine, and is clinically used for treating common cold cough, asthma and expectoration, bronchitis and asthma. The cynanchum atratum contains volatile oil, cardiac glycoside and other components, is a heat-clearing and blood-cooling medicine, and is mainly used for treating pathogenic warm, nutrient-deficiency and fever, yin-deficiency and fever, bone-steaming and fatigue fever, heat stranguria, blood stranguria and carbuncle-abscess and toxic swelling. The Laoguotou contains alkaloids and volatile oil, has effects of relieving pain, resisting inflammation, inhibiting bacteria, relieving cough and asthma, etc., and can be used for treating diseases such as lung qi stagnation, cough with excessive phlegm, chest fullness and asthma. The whole plant of Laogou head contains a substance called 7-demethoxylated tylophorine which has irreversible toxicity to the central nervous system of the animal. The radix Cynanchi Paniculati contains volatile oil, acetophenone and alkaloid, has antiviral, cardiovascular protecting, immunity regulating, antitumor, analgesic, antiinflammatory, antibacterial, blood pressure lowering, and heart rate slowing effects, and can be used for treating symptoms such as gastralgia distention, rubella, and eczema. The cynanchum atratum, the cynanchum atratum and the cynanchum paniculatum are very similar in appearance, and the phenomena of misuse, easy mixing, difficult distinction, substitution and the like frequently occur in the market, and the cynanchum atratum is also easy to mix and apply clinically, so that the medicine curative effect is affected, and the mixed use of the cynanchum atratum and the cynanchum atratum is likely to cause the medicine safety problem due to the neurotoxicity of the cynanchum atratum.
The identification of cynanchum atratum, cynomorium songaricum and cynanchum paniculatum can be performed through character identification, physicochemical identification and DNA bar code identification. Morphological identification is limited by factors such as subjective consciousness and experience of individuals, and has strong limitation. Physical and chemical identification methods such as ultraviolet spectral line group method and chemical component analysis are easy to be limited by the growth condition and experimental condition of the sample, the sensitivity of the identification methods is not high, and the result reproducibility is difficult to be ensured. DNA barcode technology cannot be directly distinguished for such closely related species, and requires joint distinction in combination with other methods. Therefore, the development of an accurate and efficient identification method is particularly important, so that effective technical support is provided for guiding clinical correct medication.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides MNP marking sites, primer compositions and kits for identifying cynanchum atratum, cynanchum atratum and cynanchum paniculatum, and the method has the characteristics of strong distinguishing force, high identification throughput and accurate result.
The invention also provides a method for identifying cynanchum atratum, cynomorium songaricum and cynanchum paniculatum.
The technical scheme for solving the technical problems is as follows:
the invention provides an MNP marking site for identifying Chinese medicinal materials including cynanchum atratum, cynomorium songaricum and cynanchum paniculatum, the MNP marking site comprises MNP-1-MNP-20 in a genome region with a plurality of nucleotide polymorphisms in cynanchum atratum, cynanchum atratum and cynanchum paniculatum.
The invention further provides a primer group based on the MNP locus and used for identifying traditional Chinese medicinal materials including cynanchum atratum, cynomorium songaricum and cynanchum paniculatum, wherein the primer group comprises 150 pairs of primers, and the nucleotide sequence of the primers is shown as SEQ ID NO. 1-300.
The invention also provides a kit for identifying cynanchum atratum, cynomorium songaricum and cynanchum paniculatum, which comprises the primer group of claim 2.
Another object of the present invention is to provide a method for identifying cynanchum atratum, cynomorium songaricum and cynanchum paniculatum, which comprises identifying cynanchum atratum, cynanchum atratum and cynanchum paniculatum by using the primer set or the kit.
Further, the method comprises the following steps:
(1) Performing multiplex PCR amplification on DNA of a sample to be detected by using the primer group to obtain a multiplex PCR amplification product, and purifying the product;
(2) Constructing a high-throughput sequencing library based on the purified multiplex PCR amplification products, obtaining a high-throughput library of a sample to be detected, and purifying the high-throughput library;
(3) Sequencing a high-throughput library of a sample to be tested to obtain sequencing data;
(4) Analyzing the sequencing data to obtain DNA fingerprint data;
(5) Comparing the DNA fingerprint data of the sample to be detected, judging the genetic similarity coefficient of the sample to be detected according to the MNP marking site, and identifying the variety of the sample to be detected according to the obtained genetic similarity coefficient.
Further, judging the variety of the sample to be detected according to the genetic similarity coefficient comprises judging that the sample to be detected and the control variety are very similar varieties or the same varieties when the genetic similarity coefficient is greater than or equal to 99%, and judging that the sample to be detected and the control variety are suspected same varieties when the genetic similarity coefficient is greater than or equal to 96%.
In the technical scheme provided by the invention, the primers of each MNP marking site comprise an upstream primer and a downstream primer, and are specifically shown in a specification table 1, wherein the upstream primer of a sequence number 1 is SEQ ID NO.1, the downstream primer of the sequence number 1 is SEQ ID NO.2, the upstream primer of the sequence number 2 is SEQ ID NO.3, the downstream primer of the sequence number 2 is SEQ ID NO.4, the upstream primer of the sequence number 3 is SEQ ID NO.5, the downstream primer of the sequence number 3 is SEQ ID NO.6, and the like.
The method for judging the varieties of the sample to be tested through the genetic similarity coefficient comprises the steps of judging that the sample to be tested and the control variety are very similar varieties or the same varieties when the genetic similarity coefficient is greater than or equal to 99%, and judging that the sample to be tested and the control variety are suspected same varieties when the genetic similarity coefficient is greater than or equal to 96%. The calculation formula of the genetic similarity is as follows:
Wherein GS is the genetic similarity coefficient of the sample to be detected and the control variety, N ij is the number of marking sites detected in the sample to be detected and the control variety, but the genotypes are not different, and N ij is the number of marking sites detected in the sample to be detected and the control variety.
The beneficial effects of the invention are as follows:
(1) The primer group provided by the invention is used for identifying cynanchum atratum, cucumis melo and cynanchum paniculatum, and sequencing amplification products by utilizing multiplex PCR amplification and fusing a second generation sequencing platform, so that the advantages of multi-target, high throughput, high efficiency and high accuracy detection of cynanchum atratum, cucumis melo and cynanchum paniculatum are realized, the primer group has very high individual distinction degree, and simultaneously has higher reproducibility and accuracy, and multiple targets can be detected once by utilizing multiplex PCR, so that the problems of false negative high and low sensitivity caused by single target amplification failure are effectively avoided;
(2) The primer group provided by the invention is used for detecting, the accuracy is high, hundreds of times of sequencing are carried out on the amplified products by matching with the second-generation high-throughput sequencer, and the output result is a base sequence, so that no parallel experiment is needed, any comparison can be carried out on the data, and the data sharing performance is strong. The kit using the primer group can have the advantages at the same time, and can effectively ensure the reproducibility and accuracy of detection, thereby ensuring the clinical medication safety and further protecting the benefits of consumers.
Drawings
FIG. 1 is a graph showing the detection number distribution of MNP marker loci of Cynanchum atratum, cynanchum otophyllum, and Cynanchum paniculatum in example 2 of the present invention;
FIG. 2 is a graph showing the MNP-marked differential ratio of Cynanchum komarovii, cynanchum otophyllum and Cynanchum paniculatum in example 2 of the present invention;
FIG. 3 is a genetic cluster map of cynanchum atratum, cynomorium venetum and cynanchum paniculatum based on MNP markers in example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.
Example 1 screening and multiplex PCR amplification primer design for identification of MNP marker loci of Cynanchum atratum, cynanchum candidum, cynanchum otophyllum and Cynanchum paniculatum
Firstly, taking cynanchum atratum as a reference genome, combining sequencing data of cynanchum atratum, cynomorium songaricum and cynanchum paniculatum, adopting Samtools (Version 1.2) and BCFtools (Version: 1.2) to carry out site polymorphism comparison analysis, and screening MNP marks according to the following principle that (1) the mark sequence is common to cynanchum atratum, cynanchum atratum and cynanchum paniculatum, but does not appear in other species, (2) the sequence has a plurality of discontinuous SNP differences, and (3) the mark sequence length is 200-300 bp. Through the screening principle, 150 MNP marker loci with high polymorphism are finally screened out.
Secondly, designing multiple PCR primer groups according to MNP labeling sites, wherein the primer design follows the principle that the primers are not interfered with each other, all the primers can be combined into a primer pool to carry out multiple PCR amplification, and finally screening out primer compositions of 150 MNP sites shown in the table 1. The primer set comprises a 1 st primer pair to a 150 th primer pair, each primer pair comprises a forward primer and a reverse primer, the nucleotide sequences of the forward primer and the reverse primer of the 1 st primer pair are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the nucleotide sequences of the forward primer and the reverse primer of the 150 th primer pair are respectively shown as SEQ ID NO. 299 and SEQ ID NO. 300. The primer set has high amplification efficiency and high identification accuracy, and meets the requirements of identifying cynanchum atratum, cynomorium atratum and cynanchum paniculatum.
TABLE 1 primer sequences corresponding to 20 MNP marker loci regions
Example 2 evaluation of MNP markers, primer compositions and kits for identification of Cynanchum atratum, cynanchum auriculatum, cynanchum otophyllum and Cynanchum paniculatum
After 150 pairs of primers were synthesized, 5ul of each primer was mixed in equal amounts to form 1:1 mixed forward and reverse primers. The detection rate, accuracy and discrimination of MNP labeling sites were tested by evaluation of MNP labeling, primers and kits developed with 2 parts of Cynanchum atratum, 2 parts of Cynanchum candidum, 2 parts of Cynanchum otophyllum and 2 parts of Cynanchum paniculatum samples provided by the unit.
In order to evaluate the above parameters, it is first necessary to obtain the MNP-labeled DNA sequence information of the above samples, and the specific experimental procedure is as follows:
Extracting DNA to obtain the DNA of the sample to be tested. Specifically, the DNA of the cynanchum atratum, cynomorium songaricum and cynanchum paniculatum are extracted by using a plant genome DNA extraction kit (manufacturer: tiangen biochemical technology (Beijing) Co., product number: DP 320), and detailed operation steps are shown in the specification of the kit. After obtaining the 8 DNA samples, 1. Mu.L of the measured concentration (using a Qubit fluorescence quantitative analyzer) was obtained, and the DNA concentrations were all in the range of 20 ng/. Mu.L to 50 ng/. Mu.L.
And amplifying MNP marker loci by multiple PCR to obtain multiple PCR amplified products. Specifically, 4. Mu.L of the primer set provided by the embodiment of the invention, 4. Mu.L of DNA of the sample to be detected (the DNA amount needs to be 200 ng) and 10. Mu. L GenoPlexs 3X T Master Mix (manufacturer: boruidi Biotechnology Co., shijia) are added into the amplification reaction of each sample to be detected, the total reaction system is 30. Mu.L, and the part less than 30. Mu.L is complemented by water. And (5) carrying out multiplex PCR amplification after shaking and mixing uniformly. Multiplex PCR amplification procedure was 95℃pre-denaturation for 3min, 95℃denaturation for 30 s,60℃annealing for 4 min, total 17 cycles, 72℃extension for 4 min. After the reaction, the PCR amplification product was stored at 4 ℃.
The PCR product was purified. The amplified DNA was purified by the magnetic bead method (manufacturer: nanjinouzan Biotech Co., ltd., product No. N411) and the specific operation was referred to the product instructions.
A high throughput sequencing library was constructed. Specifically, to the purified multiplex PCR amplification product, 10. Mu. L GenoPlexs 3 XT Master Mix, 2. Mu.L of Illumina sequencing adapter primer at a concentration of 5. Mu.M and 16. Mu.L of water were added as reagents, and the PCR reaction was performed by the following procedure, 95℃pre-denaturation for 3 min, 95℃denaturation for 15 s,60℃annealing for 15 s,70℃extension for 30 s, 8 cycles total, 72℃final extension for 5 min. After the reaction is finished, a high-throughput sequencing library of the sample to be tested is obtained.
The PCR product was purified. The high throughput sequencing library (manufacturer: nanjinouzan Biotechnology Co., ltd., product number: N411) was purified using the magnetic bead method, and the purification method was referred to the instructions of the product.
Library sequencing. Sequencing the high-throughput sequencing library by using an Illumina NextSeq1000 sequencer to obtain sequencing data of a sample to be tested, wherein the detailed sequencing steps are referred to the instruction of the sequencer, and the sequencing data are copied to a mobile hard disk after the sequencing is finished.
Sequencing data analysis. And (3) comparing the sequencing data of the samples to be tested to a reference genome of cynanchum atratum by using data comparison software Bowtie2 (version number 2.1.0), and storing the comparison result in a SAM (The Sequence Alignment/Map format) format to finally obtain the MNP marked DNA sequence of each sample to be tested. Analysis of the detection rate, accuracy and discrimination of the MNP-labeling sites can be performed by comparing these DNA base sequences.
(1) MNP marker detection rate analysis
According to the primer set provided by the embodiment 1 of the invention, multiplex PCR amplification and construction of a sequencing library are carried out, multiplex PCR amplification, second-generation high-throughput sequencing and data analysis are carried out on the DNA of the 8 samples to be detected, 143.7 MNP marks can be detected on average for each sample to be detected, the average detection rate reaches 95.8%, and the distribution of the number of detection sites of the MNP marks of the samples to be detected is shown in figure 1. The site detection ratio is not lower than 95% when the variety identification is required in national standard GB/T38551-2020, which shows that the MNP marker developed by the invention meets the requirement of the marker detection rate in variety identification application.
(2) MNP labeling accuracy analysis
The accuracy of variety identification ultimately depends on the accuracy of the genotyping of the marker loci. And calculating accuracy by using the repeatability experiment result, and further calculating the parting accuracy. The reproducibility experiment refers to two independent repeated experiments performed by different personnel, different batches of reagents and different instruments, and the accuracy refers to the proportion of marking sites with consistent typing results of the two experiments to all marking sites, and the accuracy = 1- (1-accuracy)/2.
In order to verify the accuracy of MNP labeling methods for identifying cynanchum atratum, cynanchum atratum and cynanchum paniculatum, the invention carries out reproducibility experiments on 8 samples, and as can be seen from table 2, 1150 MNP labeling sites are compared in the reproducibility experiments, and the accuracy of the MNP labeling method on typing the labeling sites is 99.57%. The high marking accuracy rate indicates that DNA fingerprint data collected by different laboratories or different times can be accurately compared with each other, and technical guarantee is provided for sharing the DNA fingerprint data.
Table 2 reproducibility of the results of typing at MNP marker loci
(3) MNP-labeling variety discrimination analysis
The 2 parts of cynanchum atratum, 2 parts of cynanchum atratum and 2 parts of cynanchum paniculatum are compared in pairs, and 28 comparison results are obtained. The proportion of MNP markers that differ between each pair of samples is referred to as the inter-sample distance, which directly reveals the ability of MNP markers to differentiate varieties. The difference MNP marking numbers of the two types of samples compared pairwise are counted, and the result shows that the average difference of 117.9 marking sites of each pair of samples is 88%, the difference proportion distribution is shown as figure 2, and the screened MNP marking polymorphism is high, so that the cynanchum atratum, cynomorium songaricum, and cynanchum paniculatum can be distinguished remarkably.
(4) MNP labeling method for variety identification of radix Cynanchi Atrati, radix Cynanchi Stauntonii, radix Cynanchi Paniculati and radix Cynanchi Paniculati
Comparing the DNA fingerprint data with a control sample to obtain a genetic similarity coefficient, and identifying the variety of the sample to be detected according to the genetic similarity coefficient. And identifying the variety of the sample to be detected according to the genetic similarity coefficient specifically comprises the step of judging that the sample to be detected and the control sample are suspected same variety when the genetic similarity coefficient is greater than or equal to 96%. The identification results of the 3 parts of cynanchum atratum, 2 parts of cynomorium venetum and 2 parts of cynanchum paniculatum provided by the Shandong province agricultural academy by using the 150 MNP marking sites and the kit provided by the embodiment of the invention are shown in Table 3.
TABLE 3 identification of species of Cynanchum atratum, cynanchum otophyllum and Cynanchum paniculatum
As is clear from Table 3, the highest genetic similarity coefficient value among the results of the pairwise comparison of 3 parts of cynanchum atratum, 2 parts of cynanchum atratum and 2 parts of cynanchum paniculatum samples is 7.81%, and the different varieties are judged. Furthermore, it was found by cluster analysis (fig. 3) that cynanchum atratum, cynomorium songaricum and cynanchum paniculatum can be classified into 4 general categories, respectively, wherein each sample under the cynanchum atratum, cynomorium songaricum and cynanchum paniculatum can be classified into one category respectively. The results show that the primer group and the identification method provided by the invention can be used for obviously distinguishing varieties among species and species, and can be used for accurately identifying cynanchum atratum, cynanchum atratum and cynanchum paniculatum, thereby having great significance in guiding clinical correct medication and normalizing market order of Chinese medicinal materials.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (3)
1. A primer group for identifying traditional Chinese medicinal materials including cynanchum atratum, cynomorium songaricum and cynanchum paniculatum is characterized in that the primer group comprises 150 pairs of primers, and the nucleotide sequences of the primers are shown as SEQ ID NO. 1-300.
2. A kit for identifying cynanchum atratum, cucumis melo and cynanchum paniculatum, comprising the primer set of claim 1.
3. A method for identifying cynanchum atratum, cynomorium songaricum and cynanchum paniculatum, comprising: identifying cynanchum atratum, cynanchum aizoon, cynomorium venetum and cynanchum paniculatum by using the primer set according to claim 1 or the kit according to claim 2.
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