SNP Molecular Markers for Identifying Angelica sinensis and Its Common Adulterated Species and Their Applications

Abstract

The present invention discloses a specific SNP molecular marker for identifying Angelica sinensis and its common adulterated species Angelica gigas, and its application. The primers corresponding to the specific SNP molecular marker are primers designed at the 9674th site in the chloroplast genome sequence NC_042826.1 with the Angelica sinensis sequence as a reference. By using the SNP molecular marker provided by the present invention in combination with the pyrosequencing method, Angelica sinensis can be accurately and rapidly distinguished from its closely related and easily adulterated species Angelica gigas with similar morphological characteristics based on a single base. The method provided by the present invention has high accuracy and strong applicability, and can be used for the quantitative detection of the adulteration amount of counterfeit products in Angelica sinensis and its processed products.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an SNP molecular marker for identifying Angelica sinensis and its common adulterated species and a quantitative detection method for its processed products. Background Art

[0002] Angelica sinensis is a large and commonly used Chinese medicinal material that is both a medicine and a food in China. It was first recorded in "Shennong Ben Cao Jing" and is the dried rhizome of the Umbelliferae plant Angelica sinensis (Oliv.) Diels. Its outer epidermis is light brown to brownish-brown, with a strong aroma. It is mainly distributed in Gansu, Sichuan, Yunnan and other places in China, and has the effects of promoting blood circulation and tonifying blood, regulating menstruation and relieving pain, and moistening the intestines and relieving constipation. Commercially available Angelica sinensis is mostly obtained through cultivation, and wild resources are extremely rare. Under the traditional cultivation mode, the incidence of pests, diseases and bolting of Angelica sinensis is relatively high, seriously affecting the yield and quality of Angelica sinensis medicinal materials and making it difficult to meet the market demand. In order to pursue profits, some merchants mix morphologically similar species into Angelica sinensis for use, resulting in uneven quality of Angelica sinensis and its derivative products, which poses a threat to the safety of consumers. Therefore, developing a detection method for the adulteration amount of adulterated 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 the integrity of the medicinal material traits for the identification of Angelica sinensis, and it is difficult to detect processed products of Angelica sinensis. Technologies such as high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are widely used in the quantitative detection and analysis of components such as ferulic acid, volatile oil and polysaccharides in Angelica sinensis. With the progress and development of modern science and technology, methods such as AFLP, ISSR, DNA barcoding, etc. are widely used in the species differentiation and identification, quality control and genetic diversity analysis of Angelica sinensis, etc., but there is a lack of a method for quantitatively detecting the adulteration ratio in Angelica sinensis and its processed products. At present, quantitative technologies such as ddPCR and VCQA have been applied to the 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 simple nucleotide polymorphisms (SNP) molecular markers being easy to scale and automate detection 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. By using pyrosequencing technology to sequence short fragments of species and detecting SNP base variations, it can be better applied to the detection of mixed samples of closely related species, and has been successfully applied to the identification of the original plants of Chinese medicinal materials such as Epimedium brevicornu, Lonicera japonica and Alisma orientale, as well as the quantitative identification of Pinellia ternata, Fritillaria cirrhosa and their closely related easily adulterated species. Therefore, it is necessary to develop a method applicable to single samples and mixed samples, and establish a molecular marker identification method for quantitative detection of Angelica sinensis and its adulterated species by detecting specific SNP sites through pyrosequencing technology. Summary of the Invention

[0004] One of the objectives 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 apply it to the adulteration qualitative detection of the medicament Angelica gigas Nakai with a similar appearance.

[0005] Another objective of the present invention is to provide the quantitative detection application of the above SNP molecular marker.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

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

[0008] The SNP markers of the present invention can distinguish the species Angelica gigas Nakai, which is easily confused with Angelica sinensis.

[0009] The SNP loci are the 9674th site (referring to the NC_042826.1 sequence) and the 38592nd site (referring to the NC_029393.1 sequence) in the chloroplast genome sequence.

[0010] More specifically, when the genotype of the 9674th site in the chloroplast genome sequence is G, the test sample contains Angelica sinensis; when the genotype of the 9674th site in the chloroplast genome sequence is A, the test sample contains an adulterated species of Angelica sinensis; when the genotype of the 38592nd site in the chloroplast genome sequence is T, the test sample contains Angelica gigas Nakai; when the genotype of the 38592nd site in the chloroplast genome sequence is C, the test sample does not contain Angelica gigas Nakai.

[0011] Among the two SNP loci, the 9674th site 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 other species are G. That is to say, at the 9674th site of the chloroplast genome sequence, Angelica sinensis has a G-to-A mutation.

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

[0013] For the above specific SNP molecular marker for identifying Angelica sinensis and its common adulterated species, the primers corresponding to the SNP molecular marker are shown in Table 1:

[0014] Table 1 Two SNP markers and specific PCR primers and sequencing primers are required for sequencing

[0015]

[0016] Note: Group 1 is the PCR primer and sequencing primer for amplifying the short fragment at the 9674th site of the chloroplast genome sequence that can be used for pyrosequencing; Group 2 is the PCR primer and sequencing primer for amplifying the short fragment where the 38592nd site of the chloroplast genome sequence that can be used for pyrosequencing is located.

[0017] Furthermore, the screening method for the two specific SNP molecular markers of the above Angelica sinensis and its closely related easily adulterated species Angelica gigas Nakai complies with the principle that there is no region with 4 or more consecutive identical bases before and after the specific SNP site, and the first 2 base sequences before the site are consistent.

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

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

[0020] (2) Based on the screening principle of SNP sites that are conserved within species and variable between species, use the downloaded chloroplast genome sequences to screen for SNP sites;

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

[0022] The present invention also discloses a method for identifying Angelica sinensis and its closely related easily adulterated species. The specific method includes the following steps:

[0023] (1) Using the genomic DNA of the test sample as a template, first perform PCR amplification on the fragment at the 9674th site of the chloroplast genome sequence for detecting whether there is an adulterated species of Angelica sinensis in the test sample to obtain an amplification product;

[0024] (2) Use the amplification product of the fragment at the 9674th site of the chloroplast genome sequence and its corresponding sequencing primer for pyrosequencing to detect the base display result at this site;

[0025] (3) When the pyrosequencing 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 Angelica sinensis components are not present in the test sample and all are adulterated species components of Angelica sinensis; when both base A and base G are shown, it can be determined that both Angelica sinensis components and adulterated species components of Angelica sinensis 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 sinensis and the types of adulterated species.

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

[0028] The present invention has discovered a specific SNP molecular marker for identifying Angelica sinensis and its closely related easily adulterated species and its application, and screened specific SNP sites of Angelica sinensis and its closely related easily adulterated species in the chloroplast genome of Angelica. Download 123 chloroplast genome sequences of Angelica from the GenBank database in NCBI. The screened SNP sites are the 9674th and 38592nd positions of the chloroplast genome sequence, and the sites have the characteristics of high specificity and strong specificity, and can be used for the identification of Angelica sinensis and its adulterated species Angelica gigas. Using the pyrosequencing technology and the 2 SNP markers screened in the present invention, it is possible to successfully identify Angelica sinensis and its common adulterated species Angelica gigas. Description of the Drawings

[0029] Figure 1 is the pyrosequencing result of the amplified product of the test sample at the 9674th position of the chloroplast genome sequence;

[0030] Figure 2 is the screening result of the specific SNP site of Angelica sinensis and the pyrosequencing result of the test sample at this specific SNP site;

[0031] Figure 3 is the pyrosequencing result of the mixed samples of Angelica sinensis and Angelica gigas powders with three different ratios in Huoxue Zhitong Powder at the 9674th position of the chloroplast genome sequence. Detailed Embodiments

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0033] Next, the present invention will be further described with specific examples in conjunction with the drawings.

[0034] Identification of Fresh Rhizomes of Angelica sinensis from Different Origins in Example 1

[0035] This example presents a method for rapidly identifying the rhizomes of Angelica sinensis from different origins, which specifically includes the following steps:

[0036] (1) Collect 4 samples of fresh rhizomes of Angelica sinensis from Lanzhou City, Dingxi City in Gansu Province, Bozhou City in Anhui Province, and Qujing City in Yunnan Province. After slicing the 4 rhizome samples and drying them at 50°C, they are crushed with a high-speed grinder, and the powder is filled into a self-sealing bag and sealed for later use.

[0037] (2) Respectively take 100 mg of the 4 rhizome powder samples and extract genomic DNA using the modified CTAB method.

[0038] (3) Using the genomic DNA of the sample to be tested as a template, first perform PCR amplification on the fragment where the 9674th site of the chloroplast genome sequence is located, using SEQ ID NO.1 and SEQ ID NO.1 amplification primers to obtain the amplification product, and detect whether there are adulterated species of Angelica sinensis in the sample to be tested;

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

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

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

[0042] (5) Use the amplification product of the fragment where the 9674th site of the chloroplast genome sequence is located and its corresponding SEQ IDNO.3 sequencing primer for pyrosequencing to detect the base display result of the 9674th site of the chloroplast genome sequence.

[0043] (6) The pyrosequencing results of the amplification products of the 4 samples at the 9674th site of the chloroplast gene sequence ( Figure 1 ) are only base G, which can determine that only Angelica sinensis components exist in the test samples, that is, the collected samples are all fresh rhizomes of Angelica sinensis.

[0044] Example 2 Identification of Angelica sinensis and Its Adulterated Species in Medicinal Material Mixed Powder

[0045] This example presents a method for rapidly identifying Angelica sinensis and its adulterated species Angelica gigas Nakai in mixed powder, which specifically includes the following steps:

[0046] (1)Collect the fresh rhizomes of Angelica sinensis and Angelica gigas, slice them respectively, dry them at 50 °C for 30 h, and then grind them into powder using a rapid grinder at a frequency of 55 HZ, rotating rapidly 4 times, 40 s each time. Put the powder into a self-sealing bag and seal it for later use.

[0047] (2)Respectively take the powder samples of Angelica sinensis and Angelica gigas, 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 the mixed samples of Angelica gigas powder and Angelica sinensis powder

[0049]

[0050] (3)Respectively take 3 powder samples mixed evenly in different proportions and extract genomic DNA using the improved CTAB method.

[0051] (4)Using the genomic DNA of the sample to be tested as a template, first amplify the fragment where the 9674th site of the CP sequence is located using the amplification primers SEQ ID NO.1 and SEQ ID NO.2 to obtain the amplification product, and detect whether there is adulterated Angelica sinensis in the sample to be tested.

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

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

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

[0055] (6)Use the amplification product of the fragment where the 9674th site of the chloroplast genome sequence is located and its corresponding SEQ ID NO.3 sequencing primer for pyrosequencing to detect the base display result of the 9674th site of the chloroplast genome sequence.

[0056] (7)The pyrosequencing results of the amplification products of the 3 mixed powder samples at the 9674th site of the chloroplast genome sequence ( Figure 2 ). When the pyrosequencing result of the amplification product at the 9674th site of the chloroplast genome sequence is only base A, it can be determined that only Angelica sinensis components exist in the test sample; when it is only base G, it can be determined that there are no Angelica sinensis components in the test sample, and it is all adulterated Angelica gigas components; when both base A and base G are shown, it can be determined that both Angelica sinensis components and adulterated Angelica sinensis components exist 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) Use the amplification product of the fragment where the 9674th site of the chloroplast genome sequence is located and its corresponding SEQ ID NO.3 sequencing primer for pyrosequencing to detect the base display result at the 9674th site of the chloroplast genome sequence.

[0069] (7) Pyrosequencing results of the amplification products of 3 mixed powder samples at the 9674th site of the chloroplast genome sequence ( Figure 3 ). The pyrosequencing result of sample 1 at the 9674th site of the chloroplast genome sequence shows that the base A is 85% and the base G is 15%, which is consistent with the expectation; the pyrosequencing result of sample 2 at the 9674th site of the chloroplast genome sequence shows that the base A is 76% and the base G is 24%, with a 1% deviation from the expected result; the pyrosequencing result of sample 3 at the 9674th site of the chloroplast genome sequence shows that the base A is 65% and the base G is 35%, which is consistent with the expectation.

[0070] Each embodiment in this specification is described in a related manner. For the same or similar parts among the embodiments, reference can be made to each other. 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 reference can be made to the relevant part of the method embodiment for the related content.

[0071] The above description is only for the preferred embodiments 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 all included in the protection scope of the present invention.

Claims

1. Application of specific SNP molecular markers in identifying Angelica sinensis and its closely related adulterated species, characterized in that, The specific SNP molecular marker is the CP9674 locus, and the CP9674 locus is the 9674th locus in the Angelica species chloroplast genome sequence NC_042826.1: when the base at the CP9674 locus is only A, the test sample is Angelica sinensis; when the base at the CP9674 locus is only G, Angelica sinensis does not exist in the test sample, and all are adulterated species of Angelica sinensis; when the base at the CP9674 locus has both A and G, both Angelica sinensis and its adulterated species exist in the test sample.

2. A method for identifying Angelica sinensis and its closely related easily adulterated species using the specific SNP molecular marker in claim 1, characterized in that, The specific SNP molecular marker is the CP9674 locus, and the method comprises the following steps: 1) Extract genomic DNA of the sample to be tested; 2) Use the forward and reverse primers SEQ ID No.1 and SEQ ID No.2 at the CP9674 locus to complete PCR amplification to obtain an amplification product; 3) Sequence with the sequencing primer SEQ ID No.3 by pyrosequencing technology to obtain the base information of the specific SNP molecular marker; 4) Determine whether the test sample is genuine Angelica sinensis or mixed with adulterated species according to the base information of the specific SNP molecular marker, specifically: when the base at the CP9674 locus of the amplification product is only A, it is determined that only Angelica sinensis components exist in the test sample; when the base at the CP9674 locus is only G, it is determined that Angelica sinensis components do not exist in the test sample, and all are adulterated species components of Angelica sinensis; when the base at the CP9674 locus shows both A and G, it is determined that both Angelica sinensis components and adulterated species components of Angelica sinensis exist in the test sample.

3. A method for quantifying Angelica sinensis in a test sample using the specific SNP molecular marker in claim 1, characterized in that: The specific SNP molecular marker is the CP9674 locus, and the method comprises the following steps: using the genomic DNA of the sample to be tested as a template, performing PCR amplification with a primer pair composed of the single-stranded DNA shown in SEQ ID No.1 in the sequence listing and the single-stranded DNA shown in SEQ ID No.2 in the sequence listing, then performing pyrosequencing with the single-stranded DNA shown in SEQ ID No.3 in the sequence listing as a primer, detecting the fluorescence signal ratio of base A at the CP9674 locus, and thereby obtaining the mass percentage of Angelica sinensis in the sample to be tested.

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