A mnp marker site, primer group, kit and identification method for identifying traditional Chinese medicinal materials radix cynanchi, radix bupleuri, old gourd head and xuchangqing

Abstract

The present application belongs to the technical field of molecular identification, and particularly relates to a MNP marker site, primer group, kit and identification method for identifying Sanguisorba officinalis, Iris lactea, Disporopsis pernyi and Disporopsis fuscopetiolata. The MNP marker site is in a genomic region with multiple nucleotide polymorphisms in Sanguisorba officinalis, Iris lactea, Disporopsis pernyi and Disporopsis fuscopetiolata, and includes MNP-1 to MNP-20. The MNP marker site provided by the present application has the advantages of a large number, high polymorphism and strong variety identification capability, and meets the needs of distinguishing similar but actually different traditional Chinese medicinal materials (Sanguisorba officinalis, Iris lactea, Disporopsis pernyi and Disporopsis fuscopetiolata). The primer group provided by the present application can obtain DNA fingerprint data of the sample to be tested, and the variety identification conclusion can be obtained by comparing the DNA fingerprint data between the samples to be tested. The detection method can compare hundreds of samples to be tested at one time, quickly obtain the variety identification conclusion, and significantly improve the accuracy and efficiency of identifying Sanguisorba officinalis, Iris lactea, Disporopsis pernyi and Disporopsis fuscopetiolata.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular identification, and particularly relates to an MNP marker site, a primer group, a kit and an identification method for identifying Radix Cynanchi, Radix Dipsaci, Radix Gynostemmatis and Radix Kuangxianqin. BACKGROUND

[0002] Radix Cynanchi, Radix Dipsaci, Radix Gynostemmatis and Radix Kuangxianqin are derived from the same family and the same genus of plants and belong to the plants of Cynanchum L. of the family Asclepiadaceae. They are very similar in appearance and are extremely easy to be confused in the process of purchase, distribution and application. Radix Dipsaci has the medicinal value of warming and resolving cold phlegm and is derived from the roots and rhizomes of the perennial herbaceous plants of Radix Dipsaci and Radix Dipsaci. Radix Cynanchi has the medicinal value of clearing and reducing virtual heat and is the roots and rhizomes of the perennial herbaceous plants of Radix Cynanchi or Radix Cynanchi. Radix Gynostemmatis has the medicinal value of relieving cough and asthma, resisting inflammation and inhibiting bacteria and is derived from the dry aboveground parts of the perennial herbaceous plant of Radix Gynostemmatis. Radix Kuangxianqin has the medicinal value of relieving pain, resisting bacteria and removing dampness and is derived from the dry roots or rhizomes of the plant of Radix Kuangxianqin.

[0003] Radix Cynanchi, Radix Dipsaci, Radix Gynostemmatis and Radix Kuangxianqin are similar in morphological characteristics, but different in medicinal ingredients and completely different in medicinal effects. Radix Dipsaci contains triterpene saponins, flavonoid glycosides and other ingredients and is a drug for reducing qi and resolving phlegm and is clinically used for the treatment of cold cough, wheezing phlegm, bronchitis and asthma. Radix Cynanchi contains volatile oil, cardiac glycosides and other ingredients and is a drug for clearing heat and cooling blood and is used for the treatment of heat caused by warm evil attacking nutrient aspect, heat due to yin deficiency, heat due to bone and exhaustion, heat stranguria, blood stranguria and carbuncle and so on. Radix Gynostemmatis contains alkaloids and volatile oil and has the effects of relieving pain, resisting inflammation and inhibiting bacteria, relieving cough and asthma and is used for the treatment of lung qi stagnation, cough with much phlegm, chest fullness and urgent dyspnea and other diseases. The whole plant of Radix Gynostemmatis contains a substance called 7-demethoxymatatabine, which has irreversible toxicity to the central nervous system of animals. Radix Kuangxianqin contains volatile oil, phenylacetone and alkaloids and has the effects of resisting virus, protecting cardiovascular, regulating immunity, resisting inflammation and inhibiting bacteria, relieving pain, resisting inflammation and inhibiting bacteria, reducing blood pressure and slowing heart rate and can be used for the treatment of stomach pain and fullness, wind rash, eczema and other symptoms. Since Radix Cynanchi, Radix Dipsaci, Radix Gynostemmatis and Radix Kuangxianqin are very similar in appearance, the phenomena of mis-picking, mis-using, easy confusion and substitution often occur in the market, and they are also easy to be confused in clinical application. This not only affects the curative effect of medication, but also may cause safety problems due to the use of Radix Gynostemmatis with neurotoxicity.

[0004] At present, the identification of Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing can be achieved by character identification, physicochemical identification and DNA barcoding identification. Morphological identification is limited by personal subjective consciousness, experience and other factors, and has strong limitations. The physicochemical identification methods such as ultraviolet spectrum line group method and chemical composition analysis are limited by the growth conditions of the samples and experimental conditions, and the sensitivity of the identification methods is not high, and the reproducibility of the results is difficult to guarantee. The DNA barcoding technology cannot directly distinguish the species with close genetic relationship, and needs to be combined with other methods for joint identification. Therefore, it is particularly important to develop an accurate and efficient identification method, so as to provide effective technical support for guiding the correct use of medicine in clinic. SUMMARY

[0005] In order to solve the problems in the prior art, the present application provides a MNP marker site, a primer composition and a kit for identifying Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing, which can distinguish between the two, and the method has the characteristics of strong distinguishing power, high identification flux and accurate results.

[0006] The present application also provides a method for identifying Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing.

[0007] The technical solutions of the present application to solve the above technical problems are as follows:

[0008] The present application provides a MNP marker site for identifying traditional Chinese medicinal materials Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing, which has multiple nucleotide polymorphisms in the genomic region of Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing, and includes MNP-1 to MNP-20.

[0009] The present application further provides a primer set based on the above MNP site for identifying traditional Chinese medicinal materials Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing, which contains 150 pairs of primers; the nucleotide sequence of the primers is shown in SEQ ID NO: 1 to SEQ ID NO: 300.

[0010] The present application also provides a kit for identifying Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing comprising the primer set of claim 2.

[0011] Another object of the present application is to provide a method for identifying Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing, which comprises using the above primer set or kit to identify Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing.

[0012] Further, the method comprises the following steps:

[0013] (1) using the primer set to perform multiplex PCR amplification on the DNA of the sample to be tested to obtain a multiplex PCR amplification product, and purifying the product;

[0014] (2) Constructing a high-throughput sequencing library based on the purified multiplex PCR amplification product, obtaining a high-throughput library of the sample to be tested, and purifying the high-throughput library;

[0015] (3) Sequencing the high-throughput library of the sample to be tested to obtain sequencing data;

[0016] (4) Analyzing the sequencing data to obtain DNA fingerprint data;

[0017] (5) Comparing the DNA fingerprint data of the sample to be tested, judging the genetic similarity coefficient of the sample to be tested according to the MNP marker site, and identifying the variety of the sample to be tested according to the obtained genetic similarity coefficient.

[0018] Further, judging the variety of the sample to be tested according to the genetic similarity coefficient, comprising: when the genetic similarity coefficient is greater than or equal to 99%, determining that the sample to be tested and the control variety are extremely similar varieties or the same variety; when the genetic similarity coefficient is greater than or equal to 96%, determining that the sample to be tested and the control sample are suspected same varieties.

[0019] In the technical scheme provided by the application, the primer of each MNP marker site comprises an upstream primer and a downstream primer, and the specific sequence is shown in Table 1 in the specification, wherein the upstream primer of No. 1 is SEQ ID NO. 1, the downstream primer of No. 1 is SEQ ID NO. 2, the upstream primer of No. 2 is SEQ ID NO. 3, the downstream primer of No. 2 is SEQ ID NO. 4, the upstream primer of No. 3 is SEQ ID NO. 5, the downstream primer of No. 3 is SEQ ID NO. 6, and so on.

[0020] When the variety of the sample to be tested is judged according to the genetic similarity coefficient provided by the application, comprising: when the genetic similarity coefficient is greater than or equal to 99%, determining that the sample to be tested and the control variety are extremely similar varieties or the same variety; when the genetic similarity coefficient is greater than or equal to 96%, determining that the sample to be tested and the control sample are suspected same varieties. The calculation formula of the genetic similarity degree is:

[0021]

[0022] Wherein, GS is the genetic similarity coefficient of the sample to be tested and the control variety, n ij is the number of marker sites detected in the sample to be tested and the control variety but without genotype difference, N ij is the number of marker sites detected in the sample to be tested and the control variety.

[0023] The beneficial effects of the application are:

[0024] (1) The primer set provided by the present application is used for identifying Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing, and the amplification products are sequenced by using multiplex PCR amplification and a second-generation sequencing platform, so that the advantages of multi-target, high-throughput, high efficiency and high accuracy detection of Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing are realized, and the primer set has high individual discrimination, and has high repeatability and accuracy, and a plurality of targets can be detected at a time by using multiplex PCR, so that the problems of high false negative rate and low sensitivity caused by amplification failure of a single target are effectively avoided.

[0025] (2) The primer set provided by the present application is used for detection, and has high accuracy, and the amplification products are sequenced hundreds of times by using a second-generation high-throughput sequencer, and the output result is a base sequence, so that parallel experiments are not needed, data can be compared at will, and data sharing is strong. The kit using the primer set can simultaneously have the above-mentioned advantages, so that the repeatability and accuracy of detection can be effectively ensured, so that the safety of clinical medication is ensured, and the interests of consumers are protected. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 2 is a distribution diagram of MNP marker sites of Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing in Example 2 of the present application;

[0027] Figure 2 Figure 3 is a distribution diagram of MNP marker differences of Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing in Example 2 of the present application;

[0028] Figure 3 Figure 4 is a genetic clustering diagram of Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing based on MNP markers in Example 2 of the present application. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present application more clear, the following will further describe the embodiments of the present application in detail.

[0030] Example 1 Screening of MNP marker sites for identifying Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing and design of multiplex PCR amplification primer

[0031] Firstly, the present application takes Cynanchum thesioides as a reference genome, combines the sequencing data of Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing, and uses Samtools (Version 1.2) and BCFtools (Version: 1.2) to perform site polymorphism comparison analysis, and the MNP markers are screened according to the following principles: (1) the marker sequence is common in Cynanchum thesioides, Reineckea carnea, Gynostemma pentaphyllum and Xu Changqing, but does not appear in other species; (2) there are multiple discontinuous SNPs on the sequence; (3) the length of the marker sequence is between 200-300 bp. Through the above screening principles, 150 MNP marker sites with high polymorphism are finally screened out.

[0032] Secondly, a multiplex PCR primer set is designed according to the above-mentioned MNP marker sites, and the primer design follows the principle of non-interference between primers. All primers can be combined into a primer pool for multiplex PCR amplification, and finally the primer composition of 150 MNP sites described in Table 1 is screened out. The primer set includes the first primer pair to the 150th primer pair, each primer pair includes a forward primer and a reverse primer, the nucleotide sequences of the forward primer and the reverse primer of the first primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and so on. The nucleotide sequences of the forward primer and the reverse primer of the 150th primer pair are shown in SEQ ID :299 and SEQ ID NO:300 respectively. The primer set has high amplification efficiency and high identification accuracy, and meets the requirements of identifying Scrophularia ningpoensis, Delphinium grandiflorum, Trichosanthes rosthornii and Adenocaulon himalaicum.

[0033] Table 1 Primer sequences corresponding to 20 MNP marker site regions

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] Example 2 Evaluation of MNP markers, primer compositions and kits for identifying Scrophularia ningpoensis, Delphinium grandiflorum, Trichosanthes rosthornii and Adenocaulon himalaicum

[0043] After the synthesis of 150 primer pairs, 5ul of each primer was mixed in equal amounts to form 1:1 mixed forward and reverse mixed primers. The MNP markers, primers and kits developed were evaluated using 2 samples of Scrophularia ningpoensis, 2 samples of Delphinium grandiflorum, 2 samples of Trichosanthes rosthornii and 2 samples of Adenocaulon himalaicum provided by the unit. The detection rate, accuracy and resolution of the MNP marker sites were tested.

[0044] In order to evaluate the above parameters, the DNA sequence information of the above samples MNP markers needs to be obtained first. The specific experimental procedure is as follows:

[0045] DNA extraction to obtain the DNA of the samples to be tested. Specifically, the DNA of the above-mentioned Radix Angelicae Pubescentis, Radix Ranunculi Ternati, old Gua and Radix Rubi Chathamiae was extracted by using a plant genomic DNA extraction kit (manufacturer: Tiangeng Biochemical Technology (Beijing) Co., Ltd., product number: DP320), and the detailed operation steps can be found in the instruction manual of the kit. After obtaining the DNA of the above-mentioned 8 samples to be tested, 1 μL of each sample was taken to determine the concentration (using a Qubit fluorescence quantifier), and the DNA concentration was within the range of 20 ng / μL-50 ng / μL.

[0046] Multiplex PCR amplification of MNP marker sites to obtain multiplex PCR amplification products. Specifically, 4 μL of the primer set provided in the embodiments of the present application, 4 μL of DNA (the amount of DNA needs to reach 200 ng) of the sample to be tested, and 10 μL of GenoPlexs 3×T Master Mix (manufacturer: Shijiazhuang Boruitai Biotechnology Co., Ltd.) were added to each sample for amplification reaction, and the total reaction system was 30 μL. If the amount is less than 30 μL, water is added to make up the difference. After oscillation and mixing, multiplex PCR amplification was performed. The multiplex PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 4 min, and a total of 17 cycles; 72℃ extension for 4 min. After the reaction was completed, the PCR amplification products were stored at 4℃.

[0047] Purification of PCR products. The amplified DNA was purified by using a magnetic bead method (manufacturer: Nanjing Nuowezan Biotechnology Co., Ltd., product number: N411), and the specific operation was referred to the instruction manual of the product.

[0048] Construction of high-throughput sequencing library. Specifically, 10 μL of GenoPlexs 3×T Master Mix, 2 μL of Illumina sequencing adapter primers with a concentration of 5 μM, and 16 μL of water were added to the purified multiplex PCR amplification products, and PCR reaction was performed according to the following program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, and 70℃ extension for 30 s, and a total of 8 cycles; 72℃ final extension for 5 min. After the reaction was completed, the high-throughput sequencing library of the sample to be tested was obtained.

[0049] Purification of PCR products. The high-throughput sequencing library was purified by using a magnetic bead method (manufacturer: Nanjing Nuowezan Biotechnology Co., Ltd., product number: N411), and the purification method was referred to the instruction manual of the product.

[0050] Library sequencing. The high-throughput sequencing library was sequenced by using an Illumina NextSeq1000 sequencer, and the sequencing data of the sample to be tested was obtained. The detailed sequencing steps can be found in the instruction manual of the sequencer, and the sequencing data was copied to a mobile hard disk after the sequencing was completed.

[0051] Sequencing data analysis. The sequencing data of the samples to be tested were aligned to the Angelica dahurica reference genome using the data alignment software Bowtie2 (version number 2.1.0), and the alignment results were saved in SAM (The Sequence Alignment / Map format) format, finally obtaining the MNP marked DNA sequence of each sample to be tested. By aligning these DNA base sequences, the detection rate, accuracy and discrimination of the MNP marker sites can be analyzed.

[0052] (1) MNP marker detection rate analysis

[0053] According to the primer set provided in Example 1 of the present application, the DNA of the 8 samples to be tested was subjected to multiplex PCR amplification, construction of sequencing library, and data analysis. The average number of MNP markers detected in each sample to be tested was 143.7, and the average detection rate was 95.8%. The distribution of the number of MNP marker detection sites of the samples to be tested is shown in Table 1. In the national standard GB / T 38551-2020, the proportion of site detection is required to be not less than 95% when identifying varieties, which shows that the MNP markers developed in the present application meet the requirements of marker detection rate in variety identification application. Figure 1

[0054] (2) MNP marker method accuracy analysis

[0055] The accuracy of variety identification ultimately depends on the accuracy of marker site genotyping. The accuracy is calculated using the reproducibility experiment results, and then the accuracy rate of genotyping is calculated. Among them, the reproducibility experiment refers to two independent repeated experiments by different personnel, different batches of reagents and different instruments, the accuracy refers to the proportion of marker sites with consistent genotyping results in two experiments to all marker sites, and the accuracy rate = 1- (1- accuracy) / 2.

[0056] In order to test the accuracy of the MNP marker method for identifying Angelica dahurica, Angelica polymorpha, old melon head and Xu Changqing, the present application carried out reproducibility experiment on the 8 samples. As can be seen from Table 2, a total of 1150 MNP marker sites were compared in the reproducibility experiment, and the accuracy rate of the MNP marker method for marker site genotyping was 99.57%. High marker accuracy indicates that the DNA fingerprint data collected by different laboratories or at different times can be accurately compared with each other, providing technical support for the sharing of DNA fingerprint data.

[0057] Table 2 Reproducibility of MNP marker site genotyping results

[0058]

[0059] (3) MNP marker method variety discrimination analysis ​

[0060] All the MNP marker genotypes of the 2 Radix Angelicae Pubescentis, 2 Radix Pulsatillae, 2 Radix Trichosanthis and 2 Radix Scrophulariae samples were compared in pairs, and 28 pairs of comparison results were obtained. The proportion of the difference in MNP markers between each pair of samples is referred to as the distance between the samples, and the distance between the samples directly shows the distinguishing ability of the MNP markers to the varieties. The number of different MNP markers in the two types of samples was counted, and the results showed that the average number of different marker sites in each pair of samples was 117.9, and the average difference proportion was 88%, and the difference proportion distribution is shown in Figure 2 The results show that the MNP marker polymorphism screened by the present application is high, and can significantly distinguish Radix Angelicae Pubescentis, Radix Pulsatillae, Radix Trichosanthis and Radix Scrophulariae.

[0061] (4) MNP marker method for variety identification of Radix Angelicae Pubescentis, Radix Pulsatillae, Radix Trichosanthis and Radix Scrophulariae

[0062] The DNA fingerprint data is compared with the control sample to obtain a genetic similarity coefficient; and the variety of the to-be-tested sample is identified according to the genetic similarity coefficient. Specifically, the variety of the to-be-tested sample is identified according to the genetic similarity coefficient as follows: when the genetic similarity coefficient is greater than or equal to 96%, it is determined that the to-be-tested sample and the control sample are suspected to be the same variety. The 150 MNP marker sites and the kit provided by the embodiments of the present application are used to perform MNP marker site difference analysis on 3 Radix Angelicae Pubescentis, 2 Radix Pulsatillae, 2 Radix Trichosanthis and 2 Radix Scrophulariae samples provided by the Shandong Academy of Agricultural Sciences, and the identification results of Radix Angelicae Pubescentis, Radix Pulsatillae, Radix Trichosanthis and Radix Scrophulariae are shown in Table 3.

[0063] Table 3 Variety identification of Radix Angelicae Pubescentis, Radix Pulsatillae, Radix Trichosanthis and Radix Scrophulariae

[0064]

[0065]

[0066] As shown in Table 3, among the results of the pairwise comparison of the 3 Radix Angelicae Pubescentis, 2 Radix Pulsatillae, 2 Radix Trichosanthis and 2 Radix Scrophulariae samples, the highest genetic similarity coefficient is 7.81%, and it is determined that they are different varieties. In addition, through cluster analysis, it is found that Figure 3 Radix Angelicae Pubescentis, Radix Pulsatillae, Radix Trichosanthis and Radix Scrophulariae can be divided into 4 categories respectively, and each sample under the Radix Angelicae Pubescentis, Radix Pulsatillae, Radix Trichosanthis and Radix Scrophulariae category can also be clustered into a category. The above results show that the primer set and the identification method provided by the present application can significantly distinguish between inter-species and intra-species varieties, and can accurately identify Radix Angelicae Pubescentis, Radix Pulsatillae, Radix Trichosanthis and Radix Scrophulariae, which has important significance for guiding the correct use of medicine in clinic and standardizing the order of traditional Chinese medicine market.

[0067] The above description is only a preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A primer set for identifying the traditional Chinese medicinal materials Baiwei, Baiqian, Laoguatou, and Xuchangqing, characterized in that, The primer set contains 150 pairs of primers; the nucleotide sequences of the primers are shown in SEQ ID NO: 1 to SEQ ID NO:

300.

2. A reagent kit for identifying Baiwei, Baiqian, Laoguatou, and Xuchangqing, characterized in that, The kit includes the primer set as described in claim 1.

3. A method for identifying Baiwei, Baiqian, Laoguatou, and Xuchangqing, characterized in that, The method includes: identifying Cynanchum paniculatum, Cynanchum paniculatum, Gynostemma pentaphyllum and Cynanchum paniculatum using the primer set of claim 1 or the kit of claim 2.

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