Primer group, kit and method for identifying cynanchum atratum and cynanchum glaucescens

Abstract

The invention provides a primer group, a kit and a method for identifying cynanchum atratum and cynanchum glaucescens, and belongs to the technical field of molecular identification. The primer group comprises a first primer pair to a 20th primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the 20th primer pair and the reverse primer of the 20th primer pair are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 40 in a sequence table. According to the primer group, the kit and the identification method provided by the invention, interspecific and intraspecific varieties can be remarkably distinguished, cynanchum atratum and cynanchum glaucescens can be accurately identified, and the primer group, the kit and the identification method have important significance in guiding clinical correct medication and standardizing the market order of traditional Chinese medicinal materials.

Description

Technical Field

[0001] The present disclosure relates to the technical field of molecular identification, and particularly relates to a primer set, a kit and a method for identifying Cynanchum atratum and Cynanchum glaucescens. Background Art

[0002] Cynanchum atratum and Cynanchum glaucescens are two traditional Chinese medicines with different functions, and some medical units are prone to confusing and misusing them. Cynanchum atratum contains volatile oils, cardiac glycosides and other components, and is a heat-clearing and blood-cooling medicine, which is mostly used for the treatment of fever caused by pathogenic warm factors injuring nutrient qi, yin deficiency fever, bone-steaming fever, postpartum blood deficiency fever, heat strangury, blood strangury, carbuncles and sores. Cynanchum glaucescens contains triterpenoid saponins, flavonoid glycosides and other components, and is a qi-descending and phlegm-resolving medicine, which is clinically mostly used for the treatment of cold cough, wheezing phlegm, bronchitis and asthma. It can be seen that the medicinal components contained in Cynanchum atratum and Cynanchum glaucescens are different, resulting in different medicinal effects. Cynanchum atratum is the root and rhizome of Cynanchum atratum Bunge or Cynanchum versicolor Bunge, which are perennial herbs of the Asclepiadaceae family. Cynanchum glaucescens comes from the root and rhizome of Cynanchum stauntonii (Decne.) Schltr. ex Levl. and Cynanchum glaucescens (Decne.) Hand.-Mazz., which are perennial herbs of the Asclepiadaceae family. Cynanchum atratum and Cynanchum glaucescens are similar in morphological characters. Due to their similar morphological characters, there are often situations of being easily confused and difficult to distinguish, miscollection, misreceiving, misuse, mixing and substitution in the market, and the situation of mixing and misusing them in medical treatment is relatively serious.

[0003] Accurately identifying medicinal plants such as Cynanchum atratum and Cynanchum glaucescens is the most basic and crucial step to ensure the quality and efficacy of drugs. At present, there are few identification studies on Cynanchum atratum and Cynanchum glaucescens, and the most commonly used method is DNA barcoding technology. DNA barcoding technology is a commonly used molecular marker technology, which uses the sequence information of one or several standard DNA fragments to identify species through homologous comparison with a reference database and phylogenetic tree analysis. The Internal Transcribed Spacer (ITS) is a relatively common DNA barcode sequence in plant identification, including the non-coding ITS1, ITS2 and the coding 5.8S region between them. The spacer region has a faster evolution rate, the non-coding region has more variable information sites, and ITS2 has a rich spatial secondary structure, which can provide additional variable information for closely related species. However, hybridization and gene insertion between species may lead to different results in phylogenetic analysis of ITS; for some closely related species, the ITS sequences may be relatively similar, and the ITS evolution rates of different species may vary greatly. Cynanchum atratum and Cynanchum glaucescens belong to the same family and genus, so it is difficult to distinguish and identify them. Therefore, there is an urgent need for a method that can accurately and efficiently identify Cynanchum atratum and Cynanchum glaucescens.

[0004] Disclosure Content

[0005] In order to solve the problems of the prior art, the embodiments of the present disclosure provide a primer set, a kit and a method for identifying Cynanchum atratum and Cynanchum glaucescens. The technical solutions are as follows:

[0006] On the one hand, the present disclosure provides a primer set for identifying Cynanchum atratum and Cynanchum stauntonii, the primer set comprising: the 1st primer pair to the 20th primer pair, each primer pair comprising a forward primer and a reverse primer, and the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primer of the 20th primer pair and the reverse primer of the 20th primer pair are shown in SEQ ID NO: 1 to SEQ ID NO: 40 in the sequence listing in sequence.

[0007] On the other hand, the present disclosure provides a kit for identifying Cynanchum atratum and Cynanchum stauntonii, the kit comprising the primer set as claimed in claim 1.

[0008] On yet another hand, the present disclosure provides a method for identifying Cynanchum atratum and Cynanchum stauntonii, the method comprising:

[0009] Performing multiplex PCR amplification on the DNA of the sample to be tested using the primer set as claimed in claim 1 to obtain a multiplex PCR amplification product;

[0010] Purifying the multiplex PCR amplification product to obtain the purified multiplex PCR amplification product;

[0011] Constructing a high-throughput sequencing library using the purified multiplex PCR amplification product to obtain a high-throughput library of the sample to be tested;

[0012] Purifying the high-throughput library to obtain the purified high-throughput library;

[0013] Sequencing the purified high-throughput library to obtain sequencing data;

[0014] Analyzing the sequencing data to obtain DNA fingerprint data;

[0015] Comparing the DNA fingerprint data with the fingerprint data of the control variety, and judging the genetic similarity coefficient of the sample to be tested according to the MNP marker locus;

[0016] Identifying the variety of the sample to be tested according to the genetic similarity coefficient.

[0017] Specifically, when the genetic similarity coefficient is greater than or equal to 96%, it is determined that the sample to be tested and the control variety are suspected to be the same variety, and the calculation formula of the genetic similarity GS is:

[0018]

[0019] In the formula, n ij is the number of marker loci with no difference in genotypes detected in both the sample to be tested and the control variety, and N ij is the number of marker loci detected in both the sample to be tested and the control variety.

[0020] Specifically, the amplification system for multiplex PCR amplification includes: the primer set, the DNA of the sample to be tested, GenoPlexs 3×T Master Mix, and water.

[0021] The beneficial effects brought by the technical solution provided by the embodiments of the present disclosure are as follows: The primer set, kit, and identification method provided by the present invention can significantly distinguish between interspecies and intraspecies varieties, and can accurately identify Cynanchum atratum and Cynanchum glaucescens, which is of great significance for guiding correct clinical medication and standardizing the order of the Chinese herbal medicine market. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1 It is a distribution diagram of the number of detected MNP markers of the sample to be tested provided by Embodiment 3 of the present disclosure;

[0024] Figure 2 It is a distribution diagram of the difference ratio of MNP markers of the sample to be tested provided by Embodiment 3 of the present disclosure;

[0025] Figure 3 It is a genetic clustering diagram of Cynanchum atratum and Cynanchum glaucescens samples based on MNP markers provided by Embodiment 3 of the present disclosure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] To make the objectives, technical solutions, and advantages of the present disclosure clearer, the following will further describe the embodiments of the present disclosure in detail with reference to the drawings.

[0027] Embodiment 1

[0028] The embodiments of the present disclosure provide a primer set for identifying Cynanchum atratum and Cynanchum glaucescens. The primer set includes: the first primer pair to the twentieth primer pair. Each primer pair includes a forward primer and a reverse primer. The forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primers of the second to twentieth primer pairs, and the reverse primers of the twentieth primer pair are shown in SEQ ID NO: 1 to SEQ ID NO: 40 in the sequence listing in sequence. The sequences of the primer set are shown in Table 1.

[0029] Table 1 is the primer sequence of MNP markers

[0030]

[0031]

[0032] Example 2

[0033] An embodiment of the present disclosure provides a kit for identifying Cynanchi atrati Radix and Cynanchi stauntonii Radix. The kit includes the primer set provided in Example 1.

[0034] Example 3

[0035] An embodiment of the present disclosure provides a method for identifying Cynanchi atrati Radix and Cynanchi stauntonii Radix. The method includes:

[0036] Performing multiplex PCR amplification on the DNA of the sample to be tested using the primer set provided in Example 1 to obtain a multiplex PCR amplification product;

[0037] Purifying the multiplex PCR amplification product to obtain a purified multiplex PCR amplification product;

[0038] Constructing a high-throughput sequencing library using the purified multiplex PCR amplification product to obtain a high-throughput library of the sample to be tested;

[0039] Purifying the high-throughput library to obtain a purified high-throughput library;

[0040] Sequencing the purified high-throughput library to obtain sequencing data;

[0041] Analyzing the sequencing data to obtain DNA fingerprint data;

[0042] Comparing the DNA fingerprint data and judging the genetic similarity coefficient of the sample to be tested according to the MNP marker locus;

[0043] Identifying the variety of the sample to be tested according to the genetic similarity coefficient.

[0044] Specifically, when the genetic similarity coefficient is greater than or equal to 96%, it is determined that the sample to be tested and the control variety are suspected to be the same variety, and the calculation formula of the genetic similarity GS is:

[0045]

[0046] In the formula, n ij is the number of marker loci with no difference in genotypes detected in both the sample to be tested and the control variety, and N ij is the number of marker loci detected in both the sample to be tested and the control variety.

[0047] In this example, 5 samples of Cynanchi atrati Radix and 5 samples of Cynanchi stauntonii Radix collected by our own laboratory were used as the samples to be tested, and the primer set provided in Example 1 was used to evaluate the detection rate, accuracy and discrimination.

[0048] Extract the DNA of the test samples. Specifically, use a plant genomic DNA extraction kit (manufacturer: Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: DP320) to extract the DNA of the above 5 Cynanchum atratum samples and the DNA of the above 5 Cynanchum stauntonii samples respectively. The detailed operation steps can be found in the instruction manual of this kit. After obtaining the DNA of the above 10 test samples, take 1 μL of each to measure the concentration of the DNA of the test samples (using a Qubit fluorescence quantifier), and the measured concentrations of the DNA of the test samples are all within the range of 20 ng / μL to 50 ng / μL.

[0049] Perform multiplex PCR amplification on the DNA of the test samples using the primer set provided in Example 1 to obtain multiplex PCR amplification products. Specifically, in the amplification reaction of each test sample, add 4 μL of the primer set provided in this example of the present invention (the concentration of each forward primer and each reverse primer is 0.2 μM), 4 μL of the DNA of the test sample (the DNA amount needs to reach 200 ng), and 10 μL of GenoPlexs 3×T Master Mix (manufacturer: Shijiazhuang Boreidy Biotechnology Co., Ltd.). The total reaction system is 30 μL, and the insufficient part is made up with water. After mixing well by oscillation, perform multiplex PCR amplification. The multiplex PCR amplification program is shown in Table 1.

[0050] Table 1 is the multiplex PCR amplification program

[0051]

[0052] After the multiplex PCR amplification reaction, store the multiplex PCR amplification products at 4°C.

[0053] Purify the multiplex PCR amplification products to obtain purified multiplex PCR amplification products. Specifically, use the magnetic bead method to purify the multiplex PCR amplification products (magnetic bead manufacturer: Nanjing Novoprotein Science and Technology Co., Ltd., product number: N411), and the specific operation refers to the product instruction manual.

[0054] Construct a high-throughput sequencing library using the purified multiplex PCR amplification products to obtain the high-throughput library of the test samples. Specifically, add the following reaction reagents to 16 μL of the purified multiplex PCR amplification products: 10 μL of GenoPlexs 3×T Master Mix and 4 μL of Illumina sequencing adapter primer with a concentration of 5 μM, and perform PCR amplification reaction according to the amplification program provided in Table 2. After the amplification reaction, obtain the high-throughput library of the test samples.

[0055] Table 2 is the PCR amplification program

[0056]

[0057] Purify the high-throughput library to obtain the purified high-throughput library. Specifically, the high-throughput library is purified by the magnetic bead method (magnetic bead manufacturer: Nanjing Novoprotein Biological Technology Co., Ltd., product number: N411), and the purification method refers to the product manual.

[0058] Sequence the purified high-throughput library to obtain sequencing data. Specifically, use the Illumina NextSeq 1000 sequencer to sequence the purified high-throughput sequencing library to obtain the sequencing data of the test sample. The detailed sequencing steps refer to the manual of this sequencer. After sequencing, copy the sequencing data to a mobile hard drive.

[0059] Analyze the sequencing data to obtain DNA fingerprint data. Use the data alignment software Bowtie2 (version number 2.1.0) to align the sequencing data of the test sample to the Cynanchum atratum reference genome. The alignment results are saved in the SAM (The Sequence Alignment / Map format), and finally obtain the DNA base sequence of the MNP markers for each test sample. By comparing these DNA base sequences, the detection rate, accuracy, and discrimination of the MNP marker sites can be analyzed.

[0060] Detection rate analysis

[0061] Perform multiplex PCR amplification and construction of sequencing libraries according to the primer sets provided in the embodiments of the present invention. Multiplex PCR amplification, second-generation high-throughput sequencing, and data analysis were performed on the DNA of these 10 test samples. On average, 19 MNP markers can be detected for each test sample, and the average detection rate reaches 95.5%. The distribution of the detected MNP marker sites in the test samples is as Figure 1 shown. In the national standard GB / T 38551-2020, it is required that the site detection ratio is not less than 95% during variety identification. It can be seen that the primer sets provided in this application can meet the requirements for the marker detection rate in variety identification applications.

[0062] Accuracy analysis

[0063] The accuracy of variety identification depends on the accuracy of marker site genotype typing. Use the results of the reproducibility experiment to calculate the precision, and then calculate the accuracy of typing. Among them, the reproducibility experiment refers to two independent repeated experiments performed by different personnel, different batches of reagents, and different instruments. The precision refers to the proportion of marker sites with consistent typing results in the two experiments. Accuracy = 1 - (1 - precision) / 2.

[0064] To test the accuracy of the MNP marker method for identifying Cynanchum atratum and Cynanchum glaucescens, the present invention conducted a reproducibility experiment on these 10 test samples. The specific results are shown in Table 3.

[0065] Table 3 Reproducibility of MNP Marker Locus Genotyping Results

[0066]

[0067]

[0068] In Table 3, MSBW and BWBZP are Cynanchum versicolor from different sources, ZLBW is Cynanchum atratum, and BQ and BQBZP are Cynanchum glaucescens from different sources.

[0069] As can be seen from Table 3, a total of 191 MNP marker loci were compared in the reproducibility test, and the accuracy rate of MNP marker method for locus genotyping was 100%. The high marker accuracy indicates that DNA fingerprint data collected by different laboratories or at different times can be accurately compared with each other, providing technical guarantee for the sharing of DNA fingerprint data.

[0070] Variety Discrimination Analysis

[0071] All MNP marker genotypes detected from the above 5 Cynanchum versicolor samples and 5 Cynanchum glaucescens samples were compared pairwise, and a total of 25 pairs of comparison results were obtained. The proportion of MNP markers with differences between each pair of samples was called the distance between samples, and the distance between samples directly showed the discrimination ability of MNP markers for varieties. The number of different MNP markers in pairwise comparison of these two types of samples was counted, as shown in Table 4 specifically.

[0072] Table 4 Variety Identification of Cynanchum versicolor and Cynanchum glaucescens

[0073]

[0074]

[0075] Combined with Table 4, it can be seen that the average difference between each pair of samples to be tested is 17.6 marker loci, and the average difference ratio is 96.58%. The distribution of the difference ratio is as Figure 2 shown. It can be known from Figure 2 that the MNP markers screened by the primer group provided in this embodiment have high polymorphism and can significantly distinguish Cynanchum versicolor and Cynanchum glaucescens.

[0076] Variety Identification of Cynanchum versicolor and Cynanchum glaucescens

[0077] Compare the DNA fingerprint data with the control variety, and judge the genetic similarity coefficient of the sample to be tested according to the MNP marker locus; identify the variety of the sample to be tested according to the genetic similarity coefficient. Specifically, when the genetic similarity coefficient is greater than or equal to 96%, it is determined that the sample to be tested and the control variety are suspected to be the same variety, and the calculation formula of the genetic similarity GS is:

[0078]

[0079] In the formula, nij The number of marker loci with no genotype difference detected in both the sample to be tested and the control variety, N ij The number of marker loci detected in both the sample to be tested and the control variety. The primer sets and kits provided in the examples of the present invention were used to analyze the differences in MNP marker loci of 4 Cynanchum atratum and 4 Cynanchum glaucescens provided by the Shandong Academy of Agricultural Sciences. The identification results of Cynanchum atratum and Cynanchum glaucescens are shown in Table 4.

[0080] As can be seen from Table 4, among the pairwise comparisons of 4 Cynanchum atratum samples and 4 Cynanchum glaucescens samples, the genetic similarity coefficient (GS value) between Cynanchum atratum -1 (ZLBW-1) and Cynanchum glaucescens -3 (BQ-3) is the highest, which is 6.25%. This GS value is much less than 96%, and they are determined to be different varieties. In addition, through cluster analysis (see Figure 3 ), 4 Cynanchum atratum and 4 Cynanchum glaucescens can be divided into two major categories, and each sample under the major categories of Cynanchum atratum and Cynanchum glaucescens can also be clustered into one category respectively. The above results show that the primer sets and identification methods provided by the present invention can significantly distinguish varieties between species and within species, and can accurately identify Cynanchum atratum and Cynanchum glaucescens, which is of great significance for guiding correct clinical medication and regulating the order of the Chinese herbal medicine market.

[0081] The above are only optional embodiments of the present disclosure, and are not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A primer set for identifying Bletilla striata and Bletilla striata, characterized in that: The primer set includes: the 1st primer pair to the 20th primer pair, each of the primer pairs includes a forward primer and a reverse primer, and the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primer of the 20th primer pair and the reverse primer of the 20th primer pair are respectively shown as SEQ ID NO: 1 to SEQ ID NO: 40 in the sequence listing.

2. A kit for identifying Bletilla striata and Bletilla striata, characterized in that: The kit comprises the primer set as claimed in claim 1.

3. A method for identifying Bletilla striata and Bletilla striata, characterized in that: The method comprises: Performing multiple PCR amplification on the DNA of the sample to be tested using the primer set as claimed in claim 1 to obtain multiple PCR amplification products; Purifying the multiplex PCR amplification products to obtain purified multiplex PCR amplification products; Constructing a high-throughput sequencing library using the purified multiplex PCR amplification products to obtain a high-throughput library of the sample to be tested; Purifying the high-throughput library to obtain the purified high-throughput library; Sequencing the purified high-throughput library to obtain sequencing data; Analyzing the sequencing data to obtain DNA fingerprint data; Comparing the DNA fingerprint data with the fingerprint data of the control variety, and determining the genetic similarity coefficient of the sample to be tested according to the MNP marker site; The variety of the sample to be tested is identified according to the genetic similarity coefficient.

4. The method according to claim 3, characterized in that When the genetic similarity coefficient is greater than or equal to 96%, the sample to be tested and the reference variety are determined to be suspected to be the same variety, and the calculation formula of the genetic similarity GS is: Where n ij N is the number of marker sites with no genotype difference detected in the sample to be tested and the control variety, ij It is the number of marker sites detected in both the sample to be tested and the control variety.

5. The method according to claim 3, characterized in that: The amplification system of the multiplex PCR amplification includes: the primer set, the DNA of the sample to be tested, GenoPlexs 3×T Master Mix and water.

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