CN110747290A - ITS2 sequence amplification universal primer, method for identifying traditional Chinese medicinal materials and kit - Google Patents
ITS2 sequence amplification universal primer, method for identifying traditional Chinese medicinal materials and kit Download PDFInfo
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Abstract
The invention discloses an ITS2 sequence amplification universal primer, wherein the forward primer sequence is shown as SEQ ID No.1, and the reverse primer sequence is shown as SEQ ID No. 2; also discloses application of the ITS2 sequence amplification universal primer in traditional Chinese medicine identification. The invention also discloses a method and a kit for identifying the traditional Chinese medicinal materials. The method overcomes the limitation of the currently adopted ITS2 barcode amplification primer ITS2F/ITS3R in universality, improves the universality of the primer by 20 percent, and has important value in the field of traditional Chinese medicine identification.
Description
Technical Field
The invention relates to the field of molecular biology detection, and particularly relates to an ITS2 sequence amplification universal primer and application thereof, and a method and a kit for identifying traditional Chinese medicinal materials.
Background
The DNA barcode molecular identification method utilizes a section of recognized and relatively short DNA sequence in a genome to identify species, has the characteristics of universality, stability, accuracy and the like, is incorporated into national pharmacopoeias of China, British, America, Japan and the like, and is mainly used for identifying medicinal plants, medicinal materials and part of decoction pieces at present. Identification of traditional Chinese medicine D N A barcode molecules is a method system for identifying traditional Chinese medicines by taking ITS2 (ribosome D NA second internal transcribed spacer) as a main barcode sequence.
An ITS2 sequence amplification primer recommended by the ' pharmacopoeia of the people's republic of China ' in the 2015 edition is ITS2F/ITS3R, the primer has certain limitation on species universality, the PCR amplification of common Chinese medicinal materials such as Chinese yam, cinnamon, rhizoma polygonati, polygonum cuspidatum and the like is difficult (Vaseline, a Chinese pharmacopoeia Chinese medicinal material DNA bar code standard sequence, scientific publishing agency), a special primer needs to be designed for the PCR amplification of the ITS2 sequence, and the primer does not meet the universality requirement of a DNA bar code molecular identification method.
Disclosure of Invention
In order to overcome the limitation of the universality of the existing ITS2 barcode amplification primer ITS2F/ITS3R, the invention provides a pair of ITS2 sequence universal primers through development, design, screening and verification, and the universality of the primers is improved by nearly 20%.
The technical scheme of the invention is as follows:
the invention provides an ITS2 sequence amplification universal primer in a first aspect, wherein the ITS2 sequence amplification universal primer has a forward primer sequence of ITS2F 1: CAGAATCCCGTGAACCATC (SEQ ID No.1), and the reverse primer sequence is ITS2R 1: TCCTCCGCTTATTGATATGC (SEQ ID No. 2).
In a second aspect, the invention provides an application of the ITS2 sequence amplification universal primer in traditional Chinese medicine identification. Preferably, the Chinese medicinal materials are plant medicinal materials.
In a third aspect, the present invention provides a method for identifying a Chinese medicinal material, comprising the steps of:
step 1, pretreating a Chinese medicinal material sample to extract genome DNA of the Chinese medicinal material sample;
step 2, taking the genomic DNA extracted in the step 1 as a template, and carrying out PCR amplification on the genomic DNA by using an ITS2 sequence amplification universal primer, wherein the forward primer sequence of the ITS2 sequence amplification universal primer is shown as SEQ ID No.1, and the reverse primer sequence is shown as SEQ ID No. 2;
and 3, detecting the PCR amplification product obtained in the step 2 by adopting an agarose gel electrophoresis method.
In a preferred embodiment, the method further comprises: the genomic DNA with PCR amplified bands was sequenced using the same sequencing primers as the ITS2 sequence amplification universal primers described above.
In a preferred embodiment, the method further comprises: and (3) carrying out sequence splicing on the sequencing result to obtain the DNA barcode sequence of the Chinese medicinal material test sample.
In a preferred embodiment, the method further comprises: BLAST identification is carried out on the DNA barcode sequence by adopting a database, and the species of the Chinese medicinal material test sample is judged.
In a preferred embodiment, the database includes the Chinese medicinal material DNA barcode identification system (http:// www.tcmbarcode.cn) and the GenBank database BLAST identification system (http:// BLAST. ncbi. nlm. nih. gov/BLAST. cgi).
In a preferred embodiment, the traditional Chinese medicinal materials are plant medicinal materials, and comprise medicinal plants such as polygonatum sibiricum which is difficult to amplify by adopting ITS2 sequence amplification primers ITS2F/ITS3R recommended by 2015 edition pharmacopoeia of the people's republic of China.
The invention also provides a kit for identifying traditional Chinese medicinal materials in a fourth aspect, the kit comprises ITS2 sequence amplification universal primers, and the forward primer sequence of the ITS2 sequence amplification universal primers is ITS2F 1: CAGAATCCCGTGAACCATC (SEQ ID No.1), and the reverse primer sequence is ITS2R 1: TCCTCCGCTTATTGATATGC (SEQ ID No. 2).
The method overcomes the limitation of the currently adopted ITS2 barcode amplification primer ITS2F/ITS3R in universality, improves the universality of the primer by nearly 20 percent, and has important value in the field of traditional Chinese medicine identification.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space. It is therefore contemplated to cover by the present invention, equivalents and modifications that fall within the scope of the invention, and that fall within the scope of the invention.
The present invention will be further described with reference to the accompanying drawings to fully illustrate the objects, technical features and technical effects of the present invention.
Drawings
FIG. 1 shows the result of primary screening agarose gel electrophoresis based on 12 medicinal plants in a preferred embodiment of the invention, wherein the 12 medicinal plants are Liliaceae, Plantaginaceae, Labiatae, Leguminosae, Compositae, Ranunculaceae, Oleaceae, Solanaceae, Umbelliferae, Pinaceae, Amaranthaceae, Convolvulaceae, respectively;
FIG. 2 is a schematic diagram showing the positions of ITS2 sequence amplification universal primers in the present invention;
FIG. 3 shows the result of agarose gel electrophoresis of PCR sequence amplification of 6 Chinese medicinal materials in the preferred embodiment of the present invention, which is from right to left: DNA molecular weight markers, CK, CK, Polygonatum kingianum, Andrographis paniculata, Aristolochia debilis, gingko, glossy privet fruit and grass nut.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 design of ITS2 sequence amplification Universal primers
1. Mass data analysis: the number of ITS sequences in GenBank has increased from about 30 to about 80 ten thousand compared to the 2010 publication published as ITS2F/ITS 3R. The inventor downloads and obtains about 80 ten thousand sequences of GenBank, carries out sorting analysis, and takes the sequences as basic data of ITS2 new primer design, and the data is more representative.
K-mer modeling analysis: the traditional method for searching for a conserved region through multi-sequence comparison to design a primer not only consumes time and labor, but also easily omits important species classification units. The inventor adopts a brand new primer design idea, firstly performs 18-25bp K-mer analysis on a 5.8SrRNA region and a 28S rRNA region, screens candidate K-mer data sets covering all clusters through modeling and frequency statistics, then performs clustering analysis on K-mers according to the similarity of 90-95% to obtain a highly conserved K-mer clustering unit, and finally performs mapping positioning on the K-mer clustering unit.
3. Designing a primer: python programming is utilized, factors such as energy values, TM values, GC contents and the like of primer dimer and hairpin structures are comprehensively evaluated, and 12 pairs of primer combinations are obtained through design.
4. Primary screening of primers: the method comprises the steps of carrying out primary screening on primers by 12 families (Liliaceae, Plantaginaceae, Labiatae, Leguminosae, Compositae, Ranunculaceae, Oleaceae, Solanaceae, Umbelliferae, Pinaceae, Amaranthaceae and Convolvulaceae) with extremely large classification span, and evaluating the amplification efficiency and the length of a target product. FIG. 1 shows the results of agarose gel electrophoresis.
5. Primer verification and evaluation: verifying and evaluating 160 representative medicinal plants collected in pharmacopoeia of the people's republic of China (part one) of the 2015 edition, and finally obtaining 1 pair of ITS2 amplification primers (shown in the attached figure 2), wherein the primer sequences are as follows:
ITS2F1 CAGAATCCCGTGAACCATC(SEQ ID No.1)
ITS2R1 TCCTCCGCTTATTGATATGC(SEQ ID No.2)
example 2 amplification of ITS2 sequence Universal primer ITS2F1/ITS2R1 PCR amplification of genomic DNA of Chinese medicinal herbs
The PCR amplification of the Chinese medicinal material genome DNA comprises the following steps:
step 1, pretreating a Chinese medicinal material sample to extract genome DNA of the Chinese medicinal material sample;
the DNA extraction uses a Tiangen plant genome DNA extraction kit (Tiangen Biotech Co., China) and modifies the operation process thereof so as to obtain high-quality DNA, and the specific operation steps are as follows:
a) about 10mg of dried leaves were weighed out, rinsed with 70% ethanol, wiped dry with absorbent paper and placed in an EP tube and ground on an MM400 ball mill (Retsch, Germany).
b) 700ul of 65 ℃ pre-heating buffer solution GP1 is taken and quickly added into a centrifuge tube filled with powder (mercaptoethanol is added into pre-heating GP1 before an experiment to ensure that the final concentration is 0.1 percent), after the mixture is quickly reversed and mixed, the centrifuge tube is placed in a 65 ℃ water bath for 2 hours, and the centrifuge tube is reversed during the water bath process to mix samples for a plurality of times.
c) Add 700ul 25: 24: 1 phenol/chloroform/isoamyl alcohol, mixed well, centrifuged at 12,000rpm (-13,400 Xg) for 5min
d) Add 700ul 24: 1 chloroform/isoamyl alcohol, mixed well and centrifuged at 12,000rpm (. about.13,400 Xg) for 5 min.
e) Adding 700ul of isopropanol (-20 ℃), mixing uniformly, and standing at-20 ℃ for 10-15 min.
f) The mixed liquid was transferred to an adsorption column CB3, centrifuged at 12,000rpm (. about.13,400 Xg) for 30sec, and the waste liquid was discarded. (the volume of the adsorption column is about 700ul, and centrifugation can be added in turn.)
g) 500ul of buffer GD (previously checked for the addition of absolute ethanol) was added to adsorption column CB3, centrifuged at 12,000rpm (. about.13,400 Xg) for 30sec, the waste liquid was discarded, and adsorption column CB3 was placed in the collection tube.
h) 600ul of the rinsing solution PW (previously used, whether or not absolute ethanol was added was checked) was added to the adsorption column CB3, and the mixture was centrifuged at 12,000rpm (. about.13,400 Xg) for 30sec to discard the waste liquid, and the adsorption column CB3 was put into a collection tube.
i) Operation 7 is repeated.
j) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000rpm (. about.13,400 Xg) for 2min, and the waste liquid was decanted. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.
k) Transferring the adsorption column CB3 into a clean centrifuge tube, hanging and dripping 100uldd H2O into the middle part of the adsorption film, standing at room temperature for 2-5 min, centrifuging at 12,000rpm (13,400 Xg) for 2min, and collecting the solution into the centrifuge tube.
Step 2, taking the genomic DNA extracted in the step 1 as a template, and carrying out PCR amplification on the genomic DNA by using an ITS2 sequence amplification universal primer, wherein the forward primer sequence of the ITS2 sequence amplification universal primer is shown as SEQ ID No.1, and the reverse primer sequence is shown as SEQ ID No. 2;
a) reaction system: 2 × Taq PCR Master Mix (Beijing Edley) 12.50 μ l, forward and reverse primers each 1 μ l, ddH2O8.5. mu.l, template DNA 2. mu.l.
b) And (3) amplification procedure: 5min at 94 ℃; 94 ℃ 30sec,56 ℃ 30sec,72 ℃ 45sec,40 cycles; 10min at 72 ℃.
And 3, detecting the PCR amplification product obtained in the step 2 by adopting an agarose gel electrophoresis method (agarose concentration is 1%, electrophoresis buffer TBE, voltage is 120V, and the time is 30 min).
In this example, the PCR amplification process will be described by taking the medicinal plant rhizoma Polygonati, which is difficult to amplify ITS2F/ITS3R, as an example.
FIG. 3 shows the results of agarose gel electrophoresis of PCR sequence amplification of six Chinese medicinal materials, Polygonatum kingianum, Andrographis paniculata, Aristolochia debilis, Ginkgo biloba, Ligustrum lucidum and plant fruit nut, all of which have been successful.
If the Chinese medicinal materials are further identified, the sequence of the genome DNA with the PCR amplification band can be determined, and the sequencing primer is the same as the ITS2 sequence amplification universal primer; and then, carrying out sequence splicing on the sequencing result, removing the primer area, and obtaining the DNA barcode sequence of the Chinese medicinal material test sample. BLAST identification is carried out on the DNA barcode sequences by adopting a database, and the species of the Chinese medicinal material test sample is judged; commonly used databases include the Chinese medicinal material DNA barcode identification system (http:// www.tcmbarcode.cn) and the GenBank database BLAST identification system (http:// BLAST. ncbi. nlm. nih. gov/BLAST. cgi).
Example 3 comparison of PCR amplification efficiency of ITS2F1/ITS2R1, a universal primer for ITS2 sequence amplification of the invention, and 2015 "pharmacopoeia of the people's republic of China" recommended primer ITS2F/ITS3R
In this example, genomic DNAs of representative medicinal plants of the 67 families were PCR-amplified using the two sets of primers ITS2F1/ITS2R1 and ITS2F/ITS3R, respectively, and the results of comparison of the amplification success rates are shown in the following table:
the results show that the PCR amplification success rate of the ITS2 sequence amplification universal primer ITS2F1/ITS2R1 provided by the invention is as high as 95.52, the primer universality is improved by 19.4% compared with ITS2F/ITS3R, and the method has important value in the field of traditional Chinese medicine identification.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Sequence listing
<110> institute of medicinal plants of academy of Chinese medical sciences; a drug audit and inspection center in Heilongjiang province; harbin market food and drug inspection and detection center
<120> ITS2 sequence amplification universal primer, method for identifying traditional Chinese medicinal materials and kit
<130>2019
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>19
<212>DNA
<213> Artificial sequence ()
<400>1
cagaatcccg tgaaccatc 19
<210>2
<211>20
<212>DNA
<213> Artificial sequence ()
<400>2
tcctccgctt attgatatgc 20
Claims (10)
1. An ITS2 sequence amplification universal primer is characterized in that the ITS2 sequence amplification universal primer is shown as SEQ ID No.1 in a forward primer sequence, and is shown as SEQ ID No.2 in a reverse primer sequence.
2. The use of the ITS2 sequence amplification universal primer of claim 1 in herbal medicine identification.
3. The use of claim 2, wherein the herbal material is a botanical drug.
4. A method for identifying a traditional Chinese medicinal material is characterized by comprising the following steps:
step 1, pretreating a Chinese medicinal material sample to extract genome DNA of the Chinese medicinal material sample;
step 2, taking the genomic DNA extracted in the step 1 as a template, and carrying out PCR amplification on the genomic DNA by using an ITS2 sequence amplification universal primer, wherein the forward primer sequence of the ITS2 sequence amplification universal primer is shown as SEQ ID No.1, and the reverse primer sequence is shown as SEQ ID No. 2;
and 3, detecting the PCR amplification product obtained in the step 2 by adopting an agarose gel electrophoresis method.
5. The method of claim 4, further comprising: and (3) sequencing the genomic DNA with the PCR amplification band, wherein a sequencing primer is the same as the ITS2 sequence amplification universal primer.
6. The method of claim 5, further comprising: and carrying out sequence splicing on the sequencing result to obtain the DNA barcode sequence of the Chinese medicinal material test sample.
7. The method of claim 6, further comprising BLAST identification of the DNA barcode sequences using a database to determine the species of the herbal test article.
8. The method according to claim 7, wherein said database comprises the chinese medicinal material DNA barcode identification system and the GenBank database BLAST identification system.
9. The method of any one of claims 4-8, wherein the herbal material is a plant-based material.
10. A kit for identifying a traditional Chinese medicinal material, wherein the kit comprises the ITS2 sequence amplification universal primer according to claim 1.
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Citations (5)
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| US5876977A (en) * | 1997-01-03 | 1999-03-02 | The Chinese University Of Hong Kong | Polymerase chain reaction-restriction fragment length polymorphism test for the authentication of traditional chinese medicines |
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| US5876977A (en) * | 1997-01-03 | 1999-03-02 | The Chinese University Of Hong Kong | Polymerase chain reaction-restriction fragment length polymorphism test for the authentication of traditional chinese medicines |
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