SNP (Single Nucleotide Polymorphism) molecular marker for identifying angelica sinensis and common adulterated species thereof and application of SNP molecular marker

Abstract

The invention discloses a specific SNP (Single Nucleotide Polymorphism) molecular marker for identifying angelica sinensis and a common adulterated species angelica koreana of the angelica sinensis and application of the specific SNP molecular marker. A primer corresponding to the specific SNP molecular marker is a primer which is designed at a 9674th site in a chloroplast genome sequence NC042826.1 by taking an angelica sinensis sequence as a reference. According to the present invention, with the combination of the SNP molecular marker and the pyrosequencing method, the angelica sinensis can be accurately and rapidly distinguished from the affinis easily-adulterated angelica koreana with the similar morphological characteristics based on the single basic group, wherein the affinis easily-adulterated angelica koreana has the similar morphological characteristics; the method provided by the invention is high in accuracy and strong in applicability, and can be used for quantitative detection of the adulteration amount of counterfeit products in angelica sinensis and processed products thereof.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a SNP molecular marker for identifying angelica and its common adulterated species and quantitative detection of processed products thereof. Background Art

[0002] Angelica sinensis is a common Chinese medicinal material with both medicinal and edible properties in my country. It was first recorded in Shennong's Herbal Classic. It is the dried rhizome of Angelica sinensis (Oliv.) Diels, a plant of the Umbelliferae family. The outer skin is light brown to brown, with a strong aroma. It is mainly distributed in Gansu, Sichuan, Yunnan and other places in my country. It has the effects of promoting blood circulation, regulating menstruation and relieving pain, and moistening the intestines and relieving constipation. Most of the commercially available Angelica sinensis is obtained through cultivation, and wild resources are very rare. Under the traditional cultivation model, the incidence of diseases and insect pests and bolting of Angelica sinensis is high, which seriously affects the yield and quality of Angelica sinensis medicinal materials and is difficult to meet market demand. In pursuit of profits, some merchants mix species with similar morphology into Angelica sinensis for use, resulting in uneven quality of Angelica sinensis and its derivative products, which poses a threat to consumer safety. Therefore, developing a detection method for the amount of mixed and counterfeit products in Angelica sinensis medicinal materials and their processed products has important application value for ensuring the quality and clinical efficacy of Angelica sinensis.

[0003] Traditional identification methods require that the properties of the medicinal material are complete, and it is difficult to detect processed products of Angelica sinensis. High performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) and other technologies are widely used in the quantitative detection and analysis of components such as ferulic acid, volatile oils and polysaccharides in Angelica sinensis. With the progress and development of modern science and technology, methods such as AFLP, ISSR, and DNA barcoding are widely used in Angelica sinensis species differentiation and identification, quality control, and genetic diversity analysis, but there is a lack of methods for quantitatively detecting the proportion of adulteration in Angelica sinensis and its processed products. At present, quantitative technologies such as ddPCR and VCQA have been used for quantitative identification of mixed species, but they are limited in the application of quantitative detection of closely related species. With the development of high-throughput sequencing technology, the advantages of single nucleotide polymorphisms (SNP) molecular markers, which are easy to scale and automate, have been reflected in the field of identification of closely related species of Chinese medicinal materials. Pyrosequencing has the advantages of real-time detection and high throughput. It uses pyrosequencing technology to sequence short fragments of species. By detecting SNP base variation, it can be well applied to the detection of mixed samples of closely related species. It has been successfully applied to the identification of the origin of Chinese herbal medicines such as Epimedium, Honeysuckle and Alisma, as well as the quantitative identification of Pinellia, Fritillaria and their closely related species that are easy to be adulterated. Therefore, it is necessary to develop a method that is applicable to single samples and mixed samples, and to establish a molecular marker identification method for the quantitative detection of Angelica and its adulterated species by detecting specific SNP sites through pyrosequencing technology. Summary of the invention

[0004] One of the purposes of the present invention is to provide a specific SNP molecular marker for Angelica sinensis and its closely related species that are easily adulterated, and to apply it to the qualitative detection of adulteration of the Korean Angelica sinensis, a medicinal material with similar appearance.

[0005] The second purpose of the present invention is to provide a quantitative detection application of the above-mentioned SNP molecular markers.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides two specific SNP markers of Angelica sinensis and its closely related species that are easy to be adulterated, and the screening sequence of the specific SNP molecular marker is 123 chloroplast genome sequences of all species of Angelica sinensis. The chloroplast genome sequences are downloaded from the GenBank database in NCBI (as of April 29, 2024).

[0008] The SNP markers described in the present invention can distinguish the species Angelica sinensis from Korean Angelica sinensis, which is easily confused with Angelica sinensis.

[0009] The SNP sites are the 9674th (with the NC_042826.1 sequence as a reference) and the 38592th (with the NC_029393.1 sequence as a reference) in the chloroplast genome sequence.

[0010] More specifically, when the genotype at the 9674th position in the chloroplast genome sequence is G, Angelica sinensis is present in the test sample; when the genotype at the 9674th position in the chloroplast genome sequence is A, Angelica sinensis adulteration is present in the test sample; when the genotype at the 38592th position in the chloroplast genome sequence is T, Korean Angelica sinensis is present in the test sample; when the genotype at the 38592th position in the chloroplast genome sequence is C, Korean Angelica sinensis is not present in the test sample.

[0011] Of the two SNP sites, the 9674th position of the chloroplast genome sequence is a specific site for identifying Angelica sinensis. The genotype of Angelica sinensis at this identification site is A, and the genotypes of the other species are G. That is to say, at the 9674th position of the chloroplast genome sequence, Angelica sinensis has undergone a mutation from G to A.

[0012] The 38592nd position of the chloroplast genome sequence is a specific site for identifying Korean Angelica. The genotype of Korean Angelica at this identification site is T, and the genotypes of other species are C. That is to say, at the 38592nd position of the chloroplast genome sequence, Korean Angelica has a mutation from C to T.

[0013] The specific SNP molecular markers for identifying angelica and its common adulterated species, and the primers corresponding to the SNP molecular markers are shown in Table 1:

[0014] Table 1 2 SNP markers and sequencing require specific PCR primers and sequencing primers

[0015]

[0016] Note: Group 1 includes PCR primers and sequencing primers for amplifying a short fragment at the 9674th site of the chloroplast genome sequence that can be used for pyrophosphate sequencing; Group 2 includes PCR primers and sequencing primers for amplifying a short fragment at the 38592th site of the chloroplast genome sequence that can be used for pyrophosphate sequencing.

[0017] Furthermore, the screening method for the two specific SNP molecular markers of Angelica sinensis and its closely related and easily adulterated species Korean Angelica sinensis complies with the requirement that there are no consecutive 4 or more identical base regions before and after the specific SNP site, and the first two base sequences of the site are consistent.

[0018] Furthermore, the above-mentioned screening method for identifying SNP sites of Angelica sinensis and its common adulterated species, the screening and verification of SNP sites comprises the following steps:

[0019] (1) Download 196 chloroplast genome sequences of all species in Angelica from the GenBank database of NCBI (https: / / www.ncbi.nlm.nih.gov / );

[0020] (2) Based on the principle of intraspecific conservation and interspecific variation in SNP site screening, the downloaded chloroplast genome sequence was used to screen SNP sites;

[0021] (3) Use the BLAST function in NCBI to determine the specificity of screening SNP sites.

[0022] The present invention also discloses a method for identifying angelica and its closely related species that are easily adulterated. The specific method comprises the following steps:

[0023] (1) using the genomic DNA of the sample to be tested as a template, firstly performing PCR amplification on the fragment at position 9674 of the chloroplast genome sequence used to detect whether there is an adulterated species of Angelica sinensis in the sample to be tested, to obtain an amplified product;

[0024] (2) The amplified product of the fragment at position 9674 of the chloroplast genome sequence and its corresponding sequencing primers were used for pyrophosphate sequencing to detect the base at this position and display the results;

[0025] (3) When the pyrophosphate sequencing result of the amplified product at the 9674th position of the chloroplast genome sequence is only base A, it can be determined that only Angelica sinensis components are present in the test sample; when it is only base G, it can be determined that no Angelica sinensis components are present in the test sample and all of them are Angelica sinensis adulterated components; when both base A and base G are shown, it can be determined that both Angelica sinensis components and Angelica sinensis adulterated components are present in the test sample.

[0026] According to the above detection steps, it is possible to effectively identify whether there are adulterated species in angelica and the type of adulterated species.

[0027] The beneficial effects of the present invention are:

[0028] The present invention has discovered a specific SNP molecular marker and its application for identifying Angelica sinensis and its closely related species that are easily adulterated, and screened specific SNP sites of Angelica sinensis and its closely related species that are easily adulterated in the chloroplast genome of Angelica sinensis. 123 chloroplast genome sequences under Angelica sinensis were downloaded from the GenBank database in NCBI. The SNP sites screened were the 9674th and 38592th sites of the chloroplast genome sequence, which have the characteristics of high specificity and strong specificity, and can be used to identify Angelica sinensis and its adulterated species Korean Angelica sinensis. Using pyrophosphate sequencing technology, using the two SNP markers screened in the present invention, Angelica sinensis and its common adulterated species Korean Angelica sinensis can be successfully identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the pyrophosphate sequencing result of the amplified product of the sample to be tested at the 9674th position of the chloroplast genome sequence;

[0030] Figure 2 The specific SNP site screening results of Angelica sinensis and the pyrophosphate sequencing results of the sample to be tested at this specific SNP site;

[0031] Figure 3 This is the result of pyrophosphate sequencing at position 9674 of the chloroplast genome sequence of mixed samples of Angelica sinensis and Korean Angelica sinensis powder in three different proportions in Huoxue Zhitong Powder. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The present invention will be further described below with reference to specific examples in conjunction with the accompanying drawings.

[0034] Example 1 Identification of fresh rhizomes of Angelica sinensis from different origins

[0035] This example provides a method for quickly identifying angelica rhizomes from different origins, which specifically includes the following steps:

[0036] (1) Four fresh rhizomes of Angelica sinensis were collected from Lanzhou and Dingxi, Gansu Province, Bozhou, Anhui Province, and Qujing, Yunnan Province. The four rhizome samples were sliced ​​and dried at 50°C, then crushed with a high-speed grinder, and the powder was put into a ziplock bag and sealed for later use.

[0037] (2) Four rhizome powder samples (100 mg each) were taken and genomic DNA was extracted using the modified CTAB method.

[0038] (3) Using the genomic DNA of the sample to be tested as a template, firstly, PCR amplification is performed on the fragment at the 9674th position of the chloroplast genome sequence using SEQ ID NO.1 and SEQ ID NO.1 amplification primers to obtain an amplification product, and then detecting whether there is an adulterated species of Angelica sinensis in the sample to be tested;

[0039] (4) PCR amplification reaction system and reaction procedure:

[0040] The total volume of the amplification reaction system was 25 μL, including 12.5 μL 2×Taq Master Mix, 1 μL each of the forward and reverse 10 μmol / L primers, 1 μL DNA template, and 9.5 μL deionized water.

[0041] The reaction procedure was pre-denaturation (95°C, 5 min), 40 cycles of denaturation (95°C, 30 s)-annealing (56°C, 30 s)-extension (72°C, 45 s), and extension (72°C, 10 min).

[0042] (5) The amplified product of the fragment at position 9674 of the chloroplast genome sequence and its corresponding sequencing primer SEQ ID NO.3 were used for pyrophosphate sequencing to detect the base at position 9674 of the chloroplast genome sequence and display the results.

[0043] (6) Pyrophosphate sequencing results of the amplified products of the four samples at position 9674 of the chloroplast gene sequence ( Figure 1 ) are all just base G, which means that only Angelica sinensis components are present in the tested samples, that is, the collected samples are all fresh Angelica sinensis rhizomes.

[0044] Example 2 Identification of Angelica sinensis and its adulterated species in mixed medicinal material powder

[0045] This embodiment proposes a method for quickly identifying Angelica sinensis and its adulterated species Korean Angelica sinensis in mixed powder, which specifically comprises the following steps:

[0046] (1) Collect fresh rhizomes of Angelica sinensis and Angelica koreana, slice them and dry them at 50°C for 30 hours, grind them into powder using a rapid grinder at a frequency of 55 Hz, and rotate them rapidly for 4 times, each time for 40 seconds. Put the powder into a ziplock bag and seal it for later use.

[0047] (2) Take the angelica powder sample and the Korean angelica powder sample respectively, and mix them into a mixed powder sample with a total weight of 100 mg. The specific mixing information is shown in Table 2:

[0048] Table 2 Information on Korean Angelica powder and Angelica powder mixed samples

[0049]

[0050] (3) Three powder samples mixed evenly in different proportions were taken and genomic DNA was extracted using the modified CTAB method.

[0051] (4) Using the genomic DNA of the sample to be tested as a template, firstly, the fragment at the 9674th position of the CP sequence was amplified by PCR using the amplification primers SEQ ID NO.1 and SEQ ID NO.2 to obtain the amplified product, and then detect whether there is an adulterated species of Angelica sinensis in the sample to be tested.

[0052] (5) PCR amplification reaction system and reaction procedure:

[0053] The total volume of the amplification reaction system was 25 μL, including 12.5 μL 2×Taq Master Mix, 1 μL each of the forward and reverse 10 μmol / L primers, 1 μL DNA template, and 9.5 μL deionized water.

[0054] The reaction procedure was pre-denaturation (95°C, 5 min), 40 cycles of denaturation (95°C, 30 s)-annealing (56°C, 30 s)-extension (72°C, 45 s), and extension (72°C, 10 min).

[0055] (6) The amplified product of the fragment at position 9674 of the chloroplast genome sequence and its corresponding sequencing primer SEQ ID NO.3 were used for pyrophosphate sequencing to detect the base at position 9674 of the chloroplast genome sequence and display the results.

[0056] (7) Pyrophosphate sequencing results of the amplified products of the three mixed powder samples at position 9674 of the chloroplast genome sequence ( Figure 2 ). When the pyrophosphate sequencing result of the amplified product at the 9674th position of the chloroplast genome sequence is only base A, it can be determined that only Angelica sinensis components are present in the test sample; when it is only base G, it can be determined that there is no Angelica sinensis component in the test sample and all are adulterated species of Korean Angelica sinensis components; when both base A and base G are shown, it can be determined that both Angelica sinensis components and Angelica sinensis adulterated species components are present in the test sample.

[0057] Example 3 Quantitative detection of the amount of counterfeit products in processed angelica products

[0058] This embodiment proposes a method for quickly identifying the amount of counterfeit products in processed angelica products, which specifically includes the following steps:

[0059] (1) A Chinese patent medicine containing angelica was selected to produce Huoxue Zhitong Powder. The components of Huoxue Zhitong Powder, angelica, Panax notoginseng, frankincense (processed), borneol, Eupolyphaga sinensis, and forged natural copper, were collected from a commercial pharmacy. The powder was ground into powder using a fast grinder at a frequency of 60 Hz and rotated rapidly for 5 times, each time for 30 seconds. The powder was placed in a ziplock bag and sealed for later use.

[0060] (2) According to the provisions of the Pharmacopoeia of the People's Republic of China (2020 edition), the weight of the ingredients was reduced in proportion and mixed to prepare Huoxue Zhitong Powder: 0.100g of Angelica sinensis, 0.020g of Panax notoginseng, 0.020g of frankincense (processed), 0.005g of borneol, 0.050g of forged natural copper, and 0.030g of Eupolyphaga sinensis. The fake Korean Angelica sinensis powder was added to the Angelica sinensis powder in three different proportions. The specific mixing information is shown in Table 3:

[0061] Table 3 Information on mixed samples of Korean Angelica powder mixed with Angelica powder in Huoxue Zhitong powder

[0062]

[0063] (3) Three powder samples mixed evenly in different proportions were taken and genomic DNA was extracted using the modified CTAB method.

[0064] (4) Using the genomic DNA of the sample to be tested as a template, firstly, the fragment at the 9674th position of the CP sequence was amplified by PCR using the amplification primers SEQ ID NO.1 and SEQ ID NO.2 to obtain the amplified product, and then detect whether there is an adulterated species of Angelica sinensis in the sample to be tested.

[0065] (5) PCR amplification reaction system and reaction procedure:

[0066] The total volume of the amplification reaction system was 25 μL, including 12.5 μL 2×Taq Master Mix, 1 μL each of the forward and reverse 10 μmol / L primers, 1 μL DNA template, and 9.5 μL deionized water.

[0067] The reaction procedure was pre-denaturation (95°C, 5 min), 40 cycles of denaturation (95°C, 30 s)-annealing (56°C, 30 s)-extension (72°C, 45 s), and extension (72°C, 10 min).

[0068] (6) The amplified product of the fragment at position 9674 of the chloroplast genome sequence and its corresponding sequencing primer SEQ ID NO.3 were used for pyrophosphate sequencing to detect the base at position 9674 of the chloroplast genome sequence and display the results.

[0069] (7) Pyrophosphate sequencing results of the amplified products of the three mixed powder samples at position 9674 of the chloroplast genome sequence ( Figure 3 ). The pyrophosphate sequencing results of sample 1 at the 9674th site of the chloroplast genome sequence showed that base A was 85% and base G was 15%, which was consistent with expectations; the pyrophosphate sequencing results of sample 2 at the 9674th site of the chloroplast genome sequence showed that base A was 76% and base G was 24%, which was 1% different from the expected result; the pyrophosphate sequencing results of sample 3 at the 9674th site of the chloroplast genome sequence showed that base A was 65% and base G was 35%, which was consistent with expectations.

[0070] Each embodiment in this specification is described in a related manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. A specific SNP molecular marker for identifying Angelica sinensis and its closely related species that are prone to adulteration, characterized in that: The primers corresponding to the SNP molecular markers are primers designed based on the 9674th site in the chloroplast genome sequence NC_042826.

1.

2. The SNP molecular marker according to claim 1, characterized in that: The closely related species that is easy to be adulterated is Angelica sinensis, and the primers corresponding to the specific SNP molecular markers corresponding to the Angelica sinensis are primers designed according to the 38592th site in the chloroplast genome sequence NC_029393.

1.

3. The SNP molecular marker according to claim 2, characterized in that: When the base at position 9674 of the chloroplast genome sequence is A, the test sample is Angelica sinensis; when the base at position 9674 of the chloroplast genome sequence is G, there is an adulterated species in Angelica sinensis; when the base at position 38592 of the chloroplast genome sequence is T, the adulterated species is Korean Angelica sinensis.

4. The SNP molecular marker according to any one of claims 1 to 3, characterized in that: The primers corresponding to the SNP molecular markers include the following:

5. A method for identifying angelica and its closely related species that are easily adulterated, characterized in that: The method comprises the following steps: 1) Extracting genomic DNA from the sample to be tested; 2) performing PCR amplification using the forward and reverse primers in claim 4 to obtain an amplified product; 3) Sequencing using sequencing primers through pyrosequencing technology to obtain the base information at the SNP molecular marker; 4) According to the base results at the SNP site, determine whether the test sample is authentic Angelica sinensis or mixed with adulterated species, specifically: When the pyrophosphate sequencing result of the amplified product at the 9674th position of the chloroplast genome sequence is only base A, it can be determined that only Angelica sinensis components are present in the test sample; when it is only base G, it can be determined that no Angelica sinensis components are present in the test sample, and all of them are Angelica sinensis adulteration components; when both base A and base G are shown, it can be determined that both Angelica sinensis components and Angelica sinensis adulteration components are present in the test sample; When the pyrophosphate sequencing result of the amplified product at the 38592th position of the chloroplast genome sequence is only base T, it can be determined that only Korean angelica components are present in the test sample; when it is only base C, it can be determined that no Korean angelica components are present in the test sample.

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