CN109750093B - Method for identifying or assisting in identifying liquorice - Google Patents
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Abstract
The invention relates to a method for identifying or assisting in identifying liquorice based on single nucleotide polymorphism markers, which comprises the steps of sequencing, wherein if the 270 th site of a chloroplast gene sequence psbA-trnH is a T basic group, the liquorice to be detected is liquorice produced in Xinjiang, and if the 270 th site of the chloroplast gene sequence psbA-trnH is a C basic group, the liquorice to be detected is liquorice produced outside Xinjiang.
Description
Technical Field
The invention relates to a method for identifying or assisting in identifying liquorice produced in Xinjiang, which can be used for identifying the liquorice produced in Xinjiang.
Background
The Glycyrrhrizae radix is Glycyrrhrizae radix of Glycyrrhiza of Leguminosae, and the Glycyrrhrizae radix is dried root and rhizome of Glycyrrhrizae radix, radix Glycyrrhizae Inflatae or radix Glycyrrhiza Glabra in Chinese pharmacopoeia. In the market, the licorice root (also called Ural licorice root) is mainly from the root, and the origin is mostly distributed in Xinjiang, Gansu, Ningxia, inner Mongolia and other provinces.
At present, in the liquorice seed market, the price difference of liquorice seeds in different production places is obvious, and the price of the liquorice seeds produced in Xinjiang is generally about half lower than that of other production places (including inner Mongolia, Gansu, Jilin and other provinces). This results in that the seeds of glycyrrhiza uralensis produced in Xinjiang are often mixed with the seeds of other producing areas for sale during the market trading and circulation process. But it is difficult to accurately distinguish the liquorice produced in Xinjiang from the aspect of the appearance of the liquorice seeds.
Molecular marker techniques such as RAPD (Random Amplified Polymorphic DNA, Random Amplified Polymorphic DNA marker), AFLP (Amplified Fragment Length Polymorphism), SSR (Simple Sequence Repeat marker), etc., have poor repeatability of results, large workload and high cost.
Single Nucleotide Polymorphism (SNP) refers to a DNA sequence polymorphism caused by a single nucleotide variation occurring at the chromosomal genome level. In the long evolution process, organisms form own specific DNA base sequences, and SNP differences between species or between varieties in different places of the same species are compared, so that the genetic relationship between the species and the bioinformatics of evolution can be known, and thus, the varieties of different species or different places of the same species are precisely identified and classified. The single nucleotide polymorphism markers can help to distinguish genetic material differences between individuals by detecting single nucleotide differences on a molecular level.
Disclosure of Invention
The invention relates to a method for identifying or assisting in identifying liquorice produced in Xinjiang based on single nucleotide polymorphism markers, which is used for rapidly and accurately identifying liquorice produced in Xinjiang by carrying out DNA extraction, PCR amplification, primer screening and bioinformatics analysis on liquorice plant samples in different producing areas by applying molecular marker technology of molecular biology.
The inventor determines the main production area of liquorice by reading a large amount of documents, carrying out field research and the like. The liquorice is mainly distributed in Xinjiang, Gansu, Ningxia, Shanxi, inner Mongolia and other places at present, and the wild and the cultivation have resource distribution. A large amount of genuine liquorice wild and cultivation samples are collected by penetrating into a liquorice main production area and communicating with local large planting households.
The DNA of the collected sample is extracted, PCR amplification is carried out by using different sequence primers, sequencing is carried out, the sequences are compared by bioinformatics analysis, and the screened sequences comprise a nuclear gene sequence ITS (Internal transcribed spacer) and ITS2(Internal transcribed spacer 2), a chloroplast gene sequence psbA-trnH (mainly psbA and tRNA-His endogenous spacer sequence), rbcL (ribose 1, 5-biphosphate carboxylase/oxidative protease subnitrile) and a matrix K.
The MEGA software is used for carrying out sequence comparison and analysis, and a primer sequence which can stably and accurately identify the glycyrrhiza uralensis produced in Xinjiang is found (at a certain site or a plurality of sites of the sequence, the bases of the glycyrrhiza uralensis produced in Xinjiang are different from those of other origin samples and are respectively consistent). Through final data comparison and screening, the psbA-trnH sequence of the chloroplast genome can identify the liquorice produced in Xinjiang from the liquorice produced in other producing areas. After sequence alignment analysis, 270 sites in the psbA-trnH sequence (the psbA-trnH sequence is an independent sequence and has fixed components, namely psbA, psbA-trnH endogenous region and trnH, although the lengths of the components are different among different species, the lengths of the psbA-trnH sequences of the same species or related species are basically consistent, and after the sequence alignment is carried out by MEGA software, the bases of each sequence are corresponding, so that each sequence is ensured to generate base mutation at the 270 sites), and regular mutant bases (Single Nucleotide Polymorphisms, SNP (SNP) mutation sites) are presented. Glycyrrhiza uralensis produced in Xinjiang has T base at the 270 site of the psbA-trnH sequence, and Glycyrrhiza uralensis produced in other places have C base at the 270 site of the psbA-trnH sequence (as shown in FIG. 1).
Based on the above, the invention provides a method for identifying or assisting in identifying liquorice produced in Xinjiang based on single nucleotide polymorphism markers, which comprises the following steps:
1) extracting DNA of the liquorice to be detected;
2) through sequencing, if the 270 th site of the chloroplast gene sequence psbA-trnH is a T base, the licorice to be detected is a licorice produced in Xinjiang, and if the 270 th site of the chloroplast gene sequence psbA-trnH is a C base, the licorice to be detected is a licorice produced in other places.
In a preferred embodiment of the present invention, a primer pair may be added to perform PCR amplification to obtain an amplification product, and then the amplification product is sequenced. Preferably, the template is PCR amplified using the fwdPA/revTH primers (fwdPA/revTH primer pair) of the psbA-trnH sequence.
In a preferred embodiment of the present invention, a primer pair may be added to perform PCR amplification to obtain an amplification product, and then the amplification product is subjected to electrophoresis detection. Preferably, the amplification product is a DNA fragment of about 200 bp.
The invention relates to a primer pair for identifying or assisting in identifying liquorice produced in Xinjiang, which can be selected from the following primers: the primer pair 1, the primer pair 2 and the primer pair 3, wherein the sequence of the primer pair 1 is shown as SEQ ID NO. 1 and SEQ ID NO. 2, the sequence of the primer pair 2 is shown as SEQ ID NO. 3 and SEQ ID NO. 4, and the sequence of the primer pair 3 is shown as SEQ ID NO. 5 and SEQ ID NO. 6.
The invention relates to a method for identifying or assisting in identifying liquorice produced in Xinjiang, which comprises the following steps of:
1) extracting DNA of the liquorice to be detected as a template;
2) adopting at least one primer pair as a PCR amplification primer to carry out PCR amplification to obtain an amplification product;
3) detecting the amplification product.
Further, the detection is at a molecular level. For example, hybridization, gene chip, PCR detection, etc. PCR detection is simple and easy, and therefore, PCR detection is preferable.
In a preferred embodiment of the invention, the primer pair is selected from the group consisting of: the primer pair 1, the primer pair 2 and the primer pair 3, wherein the sequence of the primer pair 1 is shown as SEQ ID NO. 1 and SEQ ID NO. 2, the sequence of the primer pair 2 is shown as SEQ ID NO. 3 and SEQ ID NO. 4, and the sequence of the primer pair 3 is shown as SEQ ID NO. 5 and SEQ ID NO. 6.
In a preferred embodiment of the present invention, the PCR conditions for primer pair 1 are: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min.
In a preferred embodiment of the present invention, the PCR conditions for primer pair 2 are: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min.
In a preferred embodiment of the present invention, the PCR conditions for primer pair 3 are: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 48 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min.
In a preferred embodiment of the invention, the amplification product can be detected by agarose gel electrophoresis, and if an amplification band appears in electrophoresis, the liquorice to be detected is the liquorice produced in Xinjiang. Preferably, the molecular weight of the amplification band is about 200 bp.
In a preferred embodiment of the present invention, a fluorescent dye, such as the fluorescent dye SYBR Green I, may be added to the amplified reaction system, and if the system containing the fluorescent dye is detected to generate Green fluorescence, it indicates that the detected licorice is produced in Xinjiang. Preferably, the detection of whether a system containing a fluorescent dye produces green fluorescence is performed at 365nm ultraviolet wavelength.
The invention also provides a PCR reagent for identifying the liquorice produced in Xinjiang or assisting in identifying the liquorice produced in Xinjiang, which can contain at least one primer pair, and the PCR reagent can be used for detecting whether the liquorice to be detected is the liquorice produced in Xinjiang or contains the liquorice produced in Xinjiang.
The invention also provides a kit for identifying the liquorice produced in Xinjiang or assisting in identifying the liquorice produced in Xinjiang, which can contain at least one primer pair or at least one PCR reagent. The kit provides convenience for identifying the liquorice produced in Xinjiang.
In a preferred embodiment of the present invention, the kit comprises a PCR reaction buffer, a DNA polymerase and dNTPs.
In a preferred embodiment of the invention, the kit may also contain a fluorescent dye, such as the fluorescent dye SYBRGreen I.
Drawings
FIG. 1 screenshot of software of SNP variation site of glycyrrhiza psbA-trnH sequence in different producing areas
FIG. 2 is an electrophoresis test chart of primer set 1 for identifying glycyrrhiza uralensis of different origins
FIG. 3 is an electrophoresis test chart of primer set 2 for identifying glycyrrhiza uralensis of different origins
FIG. 4 is an electrophoresis test chart of primer pair 3 for identifying glycyrrhiza uralensis of different origins
The inventive content described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, which are intended as illustrations of several aspects of the invention.
EXAMPLE 1 screening of Single nucleotide polymorphism markers
The total 302 standard liquorice wild and cultivation samples collected from main production areas of Xinjiang, Gansu, inner Mongolia, Ningxia and the like can typically represent the sample types of the current main production areas.
The details of the collected licorice standards are shown in table 1 below:
the collected samples are collected on the spot (with GPS information)
1.1 Total DNA extraction
Drying the collected licorice samples of each production place by using silica gel, and crushing and grinding the samples by using a small steel ball and ball mill (PULVERISTETE 6, FRITSCH, Germany) after the licorice samples are completely dried. The total DNA of the plant sample genome is extracted by a kit method (CTAB method, Tiangen biochemistry, DP305 kit). The method comprises the following specific steps:
1) quickly transferring the ground sample powder into a 2.0mL EP tube, adding a 65 ℃ preheated buffer solution GP 1700 mu l and mercaptoethanol 1 mu l; sealing the top end of the EP tube cap with a Parafilm sealing film, turning upside down, mixing uniformly, putting into a 65 ℃ water bath for 30min, and continuously turning over the EP tube for a plurality of times in the water bath process.
2) Add 700. mu.l chloroform: isoamyl alcohol (24:1) mixed solution, fully mixed and centrifuged at 12000rpm for 5 min.
3) The supernatant separated after centrifugation was carefully transferred to a new 2.0EP tube, and 700. mu.l of isopropyl alcohol (better low temperature effect when stored in a refrigerator at-20 ℃) was added and mixed well.
4) Transferring the uniformly mixed liquid into an adsorption column CB3 for two times, centrifuging at 12000rpm for 30s, and discarding the waste liquid in the collection tube.
5) To the adsorption column CB3, 500. mu.l of buffer GD (a predetermined volume of absolute ethanol was added before use) was added, centrifuged at 12000rpm for 30 seconds, and the waste liquid in the collection tube was discarded.
6) 700. mu.l of the rinsing solution PW was added to the adsorption column CB3 (a predetermined volume of absolute ethanol was added before use), and the column was centrifuged at 12000rpm for 30 seconds to discard the waste liquid in the collection tube.
7) And 6, repeating the step.
8) The adsorption column CB3 was put back into the collection tube, centrifuged at 12000rpm for 2min, and the waste liquid in the collection tube was discarded. The adsorption column CB3 was placed in a new 1.5mL EP tube and left at room temperature for several minutes until the residual rinse solution in the adsorption column CB3 was completely dried (ethanol smell-free as a judgment standard).
9) 100 mu.l of eluent TE is suspended and added to the middle filter membrane of the adsorption column CB3 till the middle filter membrane is placed for 3 to 5 minutes at room temperature, so that the eluent can fully elute DNA.
10) The solution was collected in a 1.5ml EP tube containing adsorption column CB3 by centrifugation at 12000rpm for 2min and stored at-20 ℃ until use.
1.2 primer screening and PCR amplification
Selecting primers, carrying out PCR amplification and electrophoresis detection on total DNA of Glycyrrhrizae radix samples in different production places,
the PCR amplification reaction system is 25 μ l, and the composition is ddH2O8.5. mu.l, 2 XTaq PCR MIX 12.5. mu.l, upstream and downstream primers 1. mu.l each, and DNA template 2. mu.l.
The primer sequences and the PCR reaction conditions are respectively as follows:
ITS2 sequence, and ITS primer pair sequences (S2F: ATGCGATACTTGGTGTGAAT, S3R: GACGCTTCTCCAGACTACAAT) are respectively shown in SEQ ID NO:7 and SEQ ID NO:8,
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, 40 cycles; extension at 72 ℃ for 10 min.
ITS sequence, primer (5F: GGAAGTAAAAGTCGTAACAAGG,4R: TCCTCCGCTTATTGATATGC), the primer pair sequence is shown as SEQ ID NO:9 and SEQ ID NO:10 respectively,
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 50 ℃ for 1min, extension at 72 ℃ for 1.5 min (3 sec increase per cycle), 30 cycles; extension at 72 ℃ for 7 minutes.
The psbA-trnH sequence, a primer (fwdPA: GTTATGCATGAACGTAATGCTC, revTH: CGCGCATGGTGGATTCACAATC C), the sequences of the primer pair are respectively shown as SEQ ID NO:11 and SEQ ID NO:12,
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1.5 min, 30 cycles; extension at 72 ℃ for 7 minutes.
The sequence of matK, a primer (KIM _3F: CGTACAGTACTTTTGTGTTTACGAG, KIM _1R: ACCCAGTCCATCTGGAA ATCTTGGTTC) of the matK, the sequences of the primer pair are respectively shown as SEQ ID NO:13 and SEQ ID NO:14,
pre-denaturation at 94 ℃ for 1 min; denaturation at 94 ℃ for 30 seconds, annealing at 52 ℃ for 20 seconds, extension at 72 ℃ for 50 seconds, 35 cycles; extension at 72 ℃ for 5 minutes.
rbcL sequence, primer (1f: ATGTCACCACAAACAGAAAC,724r: TCGCATGTACCTGCAGTAGC) of the rbcL sequence, and the sequences of the primer pair are respectively shown as SEQ ID NO:15 and SEQ ID NO:16,
pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1min, 34 cycles; extension at 72 ℃ for 7 minutes.
And (3) carrying out electrophoresis detection on PCR reaction products: preparing 1% agarose gel (0.6g agarose +60mL 0.5 XTBE buffer solution, accelerating dissolution by microwave, adding 1 mul nucleic acid dye GeneGreen, cooling to about 65 ℃, pouring into a gel box for placing a comb, placing at room temperature for 10-20 minutes, pulling out the comb after the gel is completely solidified, placing the gel into an electrophoresis tank, placing the TBE buffer solution in the electrophoresis tank without the gel, dropping a PCR product into the gel, simultaneously dropping a DNA Marker (DL2000), adjusting the voltage of an electrophoresis apparatus to 100-140V, and beginning electrophoresis for 30 minutes. After electrophoresis is finished, putting the gel into a gel imager, observing an electrophoresis strip under an ultraviolet lamp, and if each sample has a single and bright strip, indicating that PCR amplification is successful, and performing subsequent sequencing work.
1.3 sequencing and sequence comparison analysis
And (3) dropping all PCR products into agarose gel for electrophoresis, cutting and recovering the positions of the bands in the gel, and performing machine sequencing on the recovered products.
After sequencing is completed, performing sequence splicing on the returned sequence by using CodonCode aligner software to remove a low-quality region sequence; MEGA software is used for sequence comparison and analysis, and a primer sequence capable of stably and accurately identifying the liquorice produced in Xinjiang is found. Through sequence alignment analysis, the 270 th site in the psbA-trnH sequence shows regular mutant bases (Single Nucleotide Polymorphisms, SNP variable sites). Glycyrrhiza uralensis from other origins have C bases at the 270 position of the psbA-trnH sequence, while Glycyrrhiza uralensis from Xinjiang have T bases at the 270 position of the psbA-trnH sequence, as shown in FIG. 1.
Example 2 identification of Glycyrrhiza uralensis Fisch.A. produced in Xinjiang based on single nucleotide polymorphism markers
2.1 Total DNA extraction
Taking a dried sample of the liquorice to be detected, directly weighing about 30mg after removing surface pollutants, putting the dried sample into a sterilized 1.5mL centrifugal tube, adding two sterilized small steel balls, and putting the sterilized small steel balls into a ball mill for grinding at 1500rpm for 3-5 minutes; weighing about 0.5g of fresh sample, removing surface pollutants, adding into a clean mortar, fully grinding into powder by using liquid nitrogen, and adding into a 1.5mL centrifugal tube in a proper amount;
adding 250 μ l of cell nucleus lysate (mainly used for cracking cell walls in a sample and releasing genetic materials in cells) and 5 μ l of RNase solution, and sufficiently shaking and uniformly mixing;
adding 20 mul of proteinase K, fully shaking and uniformly mixing;
placing in a water bath at 65 deg.C for 1 hr (repeatedly mixing under reverse rotation for several times);
adding 250 mul of lysis buffer solution, fully and uniformly mixing, completely transferring to a purification adsorption column, and centrifuging at 12000rpm for 5 minutes;
removing waste liquid in the collecting tube, adding 700 μ l of rinsing liquid (absolute ethanol is added before use), and centrifuging at 12000rpm for 40 s;
removing the waste liquid in the collecting pipe, and repeating the step 6) for 2 times;
removing the waste liquid in the collecting tube, centrifuging at 12000rpm for 2min in an empty tube, placing the purification adsorption column into a new 1.5mL centrifuge tube, and standing at room temperature for 5min until the ethanol is completely reacted;
suspending 100 μ l of sterilized elution buffer solution on the central filter membrane of the purification adsorption column, standing at room temperature for 2 minutes, centrifuging at 12000rpm for 2 minutes to obtain a sample DNA template, and storing at-20 ℃ for later use.
2.2 PCR amplification
Preparing 25 mul of PCR reaction system, placing the PCR reaction system into a 0.2ml PCR tube, wherein the system comprises 12.5 mul of 2 xTaq PCR Mix, 2 mul of DNA template, 1 mul of each primer (upstream/downstream, 2.5 mu M) and 8.5 mul of sterilized double distilled water, and carrying out PCR amplification and electrophoresis detection on the total DNA of the licorice samples to be detected in different producing areas.
The primer sequence and the PCR reaction conditions are respectively as follows:
the psbA-trnH sequence, its primer (fwdPA: GTTATGCATGAACGTAATGCTC, revTH: CGCGCATGGTGGATTCACAATCC),
pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1.5 min, 30 cycles; extension at 72 ℃ for 7 minutes.
2.3 sequencing analysis
And (3) dropping the PCR product into agarose gel for electrophoresis, cutting and recovering the position of a strip in the gel, and performing machine sequencing on the recovered product.
If the 270 th site of the psbA-trnH sequence is a T basic group, the to-be-detected liquorice is the liquorice produced in Xinjiang; if the 270 th site of the psbA-trnH sequence is C basic group, the licorice to be detected is licorice in other producing areas.
Example 3 specific primers
According to the stable SNP variation site which can stably identify the liquorice produced in Xinjiang, the specific primer for identifying the liquorice produced in Xinjiang is designed. A complementary sequence with the length of 20bp is selected at the upstream 30-49bp of the psbA-trnH sequence as an upstream primer sequence, for example, liquorice produced in Xinjiang, the 270 site of the psbA-trnH sequence is T base, three groups of primers are respectively designed around the 270 site of the psbA-trnH sequence as downstream primers, and the three groups of primers are respectively 250-bp, 260-bp, 280-bp and 270-291-bp. The reverse complementary sequences of the three sequences respectively correspond to the downstream three groups of primer sequences of the specific primer. The process can realize conversion of sequences and primer sequences through software primer 6.0, or can be manually modified.
The three primer pairs are respectively as follows:
the sequence of the primer pair 1 is shown as SEQ ID NO. 1 (5'-CTTCGAGGTAGATATTTACC-3') and SEQ ID NO. 2 (5'-GCGCGTCTTCTAAAATACGAG-3');
the sequence of the primer pair 2 is shown as SEQ ID NO. 3 (5'-CTTCGAGGTAGATATTTACC-3') and SEQ ID NO. 4 (5'-TTAAGTTCTTGCGCGTCTTCT-3');
the sequences of primer pair 3 are shown in SEQ ID NO. 5 (5'-CTTCGAGGTAGATATTTACC-3') and SEQ ID NO. 6 (5'-TTTCTTCTCTTTTAAGTTCTTG-3').
Primer 6.0 software is used for analyzing primer sequences, the annealing temperature is determined, the test optimization is carried out, and finally the PCR reaction program of each primer pair is determined as follows:
the PCR conditions for primer pair 1 were: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min at 72 ℃;
the PCR conditions for primer pair 2 were: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min at 72 ℃;
the PCR conditions for primer pair 3 were: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 48 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min.
Example 4 method for identifying glycyrrhiza uralensis produced in Xinjiang by specific primer pair
The identification system for specifically identifying the liquorice produced in Xinjiang mainly comprises a DNA extraction part, an amplification part containing a PCR reagent and an agarose gel electrophoresis detection part, wherein the PCR reagent contains a specific primer. The DNA extraction part mainly comprises cell nucleus lysate, lysate buffer solution, RNase solution, protease K, rinsing solution (absolute ethyl alcohol is not required to be added when the DNA extraction part is used), sterile elution buffer solution, a purification adsorption column, a collection tube, a 1.5mL sterilization centrifuge tube and the like. The PCR amplification part mainly comprises 2 XTaq PCR Mix, specific primers (upstream/downstream), standard Xinjiang-produced liquorice DNA (positive control), standard other-producing-place liquorice DNA (negative control), sterilized double-distilled water and the like. The agarose gel electrophoresis detection part comprises agarose, 50 XTBE buffer, DNA marker (DNA molecular weight marker) and nucleic acid dye.
The detection method comprises the following steps:
4.1 Total DNA extraction
Taking a dried sample of the liquorice to be detected, directly weighing about 30mg after removing surface pollutants, putting the weighed sample into a sterilized 1.5mL centrifugal tube, adding two sterilized small steel balls, and putting the sterilized small steel balls into a ball mill for grinding at 1500rpm for 3-5 minutes; weighing about 0.5g of fresh sample, removing surface pollutants, adding into a clean mortar, fully grinding into powder by using liquid nitrogen, and adding into a 1.5mL centrifugal tube in a proper amount;
adding 250 μ l of cell nucleus lysate (mainly used for cracking cell walls in a sample and releasing genetic materials in cells) and 5 μ l of RNase solution, and sufficiently shaking and uniformly mixing;
adding 20 mul of proteinase K, fully shaking and uniformly mixing;
placing in a water bath at 65 deg.C for 1 hr (repeatedly mixing under reverse rotation for several times);
adding 250 mul of lysis buffer solution, fully and uniformly mixing, completely transferring to a purification adsorption column, and centrifuging at 12000rpm for 5 minutes;
removing waste liquid in the collecting tube, adding 700 μ l of rinsing liquid (absolute ethanol is added before use), and centrifuging at 12000rpm for 40 s;
removing the waste liquid in the collecting pipe, and repeating the step 6) for 2 times;
removing the waste liquid in the collecting tube, centrifuging at 12000rpm for 2min in an empty tube, placing the purification adsorption column into a new 1.5mL centrifuge tube, and standing at room temperature for 5min until the ethanol is completely reacted;
suspending 100 μ l of sterilized elution buffer solution on the central filter membrane of the purification adsorption column, standing at room temperature for 2 minutes, centrifuging at 12000rpm for 2 minutes to obtain a sample DNA template, and storing at-20 ℃ for later use.
4.2 PCR amplification
Prepare 25 ul of PCR reaction system, put into 0.2ml PCR tube, the system includes 12.5 ul 2 XTaq PCR Mix, 2 ul DNA template, 1 ul each of specific primers (up/down stream, 2.5 uM), 8.5 ul of sterilized double distilled water. In addition to the sample DNA template, a positive control, a negative control and a blank control sample PCR reaction system are additionally configured, the method is the same as the above, except that the DNA template is different, and the blank control replaces the DNA template by the sterilized double distilled water.
The system solution was mixed well, centrifuged in a microcentrifuge for several seconds, and then placed in a PCR instrument.
The PCR reaction program corresponds to the specific primers and respectively comprises the following steps:
the PCR conditions for primer pair 1 were: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min at 72 ℃;
the PCR conditions for primer pair 2 were: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min at 72 ℃;
the PCR conditions for primer pair 3 were: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 48 ℃ for 1min, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min.
After the PCR reaction is finished, taking out the PCR product and storing at-4 ℃ for later use, or immediately carrying out agarose gel electrophoresis detection
4.3 electrophoretic detection
1) Agarose gel preparation
Weighing 0.6g of agarose, adding the agarose into 60mL of 0.5 xTBE buffer solution (50 xTBE buffer solution in a reagent box is suitable for long-term storage, can be directly diluted to 0.5 xTBE buffer solution by distilled water when in use, and is recommended to be prepared immediately), adding the agarose into a microwave oven to heat an accelerating solution (1-2 minutes by high fire), adding 1 mu l of nucleic acid dye when the temperature is reduced to about 65 ℃ after the agarose is completely dissolved, slowly shaking uniformly, pouring the agarose into a rubber box inserted with a comb, and solidifying at room temperature.
2) Electrophoretic detection
Adding fresh 0.5 XTBE buffer solution into an electrophoresis tank of an electrophoresis apparatus, putting the solidified gel into the electrophoresis tank (the buffer solution does not need to be over the gel), and putting one side of a sample adding hole of the gel on a negative electrode of the electrophoresis tank. Adding 5 mul of DNA marker, positive control, negative control, blank control and detection sample DNA PCR product from one end of the sample adding hole in sequence, and performing electrophoresis for 30 minutes at a constant voltage of 5V/cm.
3) Gel imaging system analysis
And after the electrophoresis is finished, putting the agarose gel into a gel imager for observation and photographing. And continuously exposing by using an ultraviolet lamp until the strips on the gel picture are clear, storing the electrophoresis gel picture result as an electronic file, and observing and recording the electrophoresis detection result.
4) Analysis of detection results
If no strip appears in the negative control and the blank control, an amplification strip of about 200bp appears in the positive control, and if a corresponding amplification strip of about 200bp appears in the sample to be detected, the sample to be detected can be judged to be a positive result (liquorice produced in Xinjiang); if the sample to be detected does not have an amplification band of about 200bp corresponding to the positive control, the sample to be detected can be judged not to be the liquorice produced in Xinjiang.
Three pairs of specific primers are used for carrying out PCR amplification and agarose gel electrophoresis detection on standard samples of liquorice in Xinjiang and other producing areas, and the three pairs of specific primers related to the invention can specifically amplify the liquorice sample produced in Xinjiang but can not amplify the liquorice samples produced in other producing areas. The electrophoresis detection maps of the three pairs of specific primers for amplifying specific licorice sample are shown in fig. 2, fig. 3 and fig. 4, the electrophoresis detection of the licorice standard product produced in Xinjiang in example 1 shows a corresponding amplified band of about 200bp, and the electrophoresis detection of the licorice standard product produced in other places in example 1 does not show a corresponding amplified band of about 200 bp. Therefore, the three pairs of specific primer pairs can quickly and specifically detect whether the liquorice to be detected is the liquorice produced in Xinjiang.
SEQUENCE LISTING
<110> Chinese herbal medicine Co., Ltd
<120> method for identifying or assisting in identifying licorice
<130>
<160> 16
<170> PatentIn version 3.5
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<213> Artificial sequence
<400> 5
cttcgaggta gatatttacc 20
<210> 6
<211> 22
<212> DNA
<213> Artificial sequence
<400> 6
tttcttctct tttaagttct tg 22
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
atgcgatact tggtgtgaat 20
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence
<400> 8
gacgcttctc cagactacaa t 21
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence
<400> 9
ggaagtaaaa gtcgtaacaa gg 22
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence
<400> 10
tcctccgctt attgatatgc 20
<210> 11
<211> 22
<212> DNA
<213> Artificial sequence
<400> 11
gttatgcatg aacgtaatgc tc 22
<210> 12
<211> 23
<212> DNA
<213> Artificial sequence
<400> 12
cgcgcatggt ggattcacaa tcc 23
<210> 13
<211> 25
<212> DNA
<213> Artificial sequence
<400> 13
cgtacagtac ttttgtgttt acgag 25
<210> 14
<211> 27
<212> DNA
<213> Artificial sequence
<400> 14
acccagtcca tctggaaatc ttggttc 27
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence
<400> 15
atgtcaccac aaacagaaac 20
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence
<400> 16
tcgcatgtac ctgcagtagc 20
Claims (8)
1. A method for identifying or assisting in identifying glycyrrhiza uralensis based on single nucleotide polymorphism markers comprises the following steps:
1) extracting DNA of glycyrrhiza uralensis to be detected as a template;
2) and adding a primer pair for PCR amplification, detecting that the 270 th site of the chloroplast gene sequence psbA-trnH is a T base, so that the Ural licorice to be detected is a licorice produced in Xinjiang, and detecting that the 270 th site of the chloroplast gene sequence psbA-trnH is a C base, so that the Ural licorice to be detected is a licorice produced outside Xinjiang.
2. The method of claim 1, further comprising the steps of: sequencing the amplified product.
3. The method of claim 1, further comprising the steps of: detecting the amplified product by agarose gel electrophoresis.
4. The method according to claim 1 or 3, wherein the primer pair is selected from at least one of primer pair 1, primer pair 2 and primer pair 3, wherein the sequence of primer pair 1 is shown as SEQ ID NO 1 and SEQ ID NO 2, the sequence of primer pair 2 is shown as SEQ ID NO 3 and SEQ ID NO 4, and the sequence of primer pair 3 is shown as SEQ ID NO 5 and SEQ ID NO 6.
5. The method of claim 3, wherein if an amplified band appears in the electrophoretic display, it indicates that the Ural licorice to be tested is Ural licorice produced in Xinjiang.
6. The method of claim 5, wherein the molecular weight of said amplified band is 200 bp.
7. The method according to any one of claims 1 to 3 and 5 to 6, wherein the DNA extraction method is CTAB method.
8. The method according to any one of claims 1 to 3 and 5 to 6, wherein the buffer for DNA extraction is isopropanol.
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| CN201810745808.0A CN109750093B (en) | 2018-07-09 | 2018-07-09 | Method for identifying or assisting in identifying liquorice |
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| CN112143826B (en) * | 2020-09-29 | 2022-06-07 | 中国中药有限公司 | SSR molecular marker primer for identifying liquorice producing area, method and application |
| CN112322781B (en) * | 2021-01-05 | 2021-04-20 | 中国中药有限公司 | SNP molecular marker for identifying liquorice produced in Gansu province and method and application thereof |
| CN112322779B (en) * | 2021-01-05 | 2021-04-16 | 中国中药有限公司 | SNP molecular marker for identifying liquorice produced in Xinjiang, and method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101216465A (en) * | 2007-12-28 | 2008-07-09 | 北京联合大学生物化学工程学院 | Licorice medicinal materials fingerprint establishment method and its standard fingerprint |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101216465A (en) * | 2007-12-28 | 2008-07-09 | 北京联合大学生物化学工程学院 | Licorice medicinal materials fingerprint establishment method and its standard fingerprint |
Non-Patent Citations (1)
| Title |
|---|
| 杨瑞.《中国药典》所载甘草的分子鉴定及市售甘草药材的质量评价.《中国优秀硕士学位论文全文数据库 医药卫生科技辑》.2017,(第08期), * |
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