CN109735644A - Identify or assisting in the primer pair and its application for identifying Radix Glycyrrhizae - Google Patents
Identify or assisting in the primer pair and its application for identifying Radix Glycyrrhizae Download PDFInfo
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Abstract
The present invention relates to the primer pair and its application for identifying Radix Glycyrrhizae is identified or assisting in, whether which can be used for being that Xinjiang production Radix Glycyrrhizae identifies to Radix Glycyrrhizae to be measured.
Description
Technical field
The present invention relates to the primer pair and its application for identifying Radix Glycyrrhizae is identified or assisting in, which can be used for Radix Glycyrrhizae to be measured
It whether is that Xinjiang production Radix Glycyrrhizae is identified.
Background technique
Radix Glycyrrhizae be pulse family Glycyrrhiza medicinal material, " Chinese Pharmacopoeia " provide licorice medicinal materials plant origin be Radix Glycyrrhizae, swollen fruit Radix or
The dry root and rhizome of glycyrrhiza glabra.For licorice medicinal materials based on Radix Glycyrrhizae (also known as Glycyrrhiza Uralensis) source, the place of production is more in the market
It is distributed in the provinces such as Xinjiang, Gansu, Ningxia, the Inner Mongol.
At present Glycyrrhiza Seeds in the market, the seed price significant difference of different sources Radix Glycyrrhizae, Xinjiang produce Radix Glycyrrhizae seed
The universal nearly half lower than other places of production (including the provinces such as Inner Mongol, Gansu, Jilin) or so of price.This result in marketing and
In the process of circulation, the seed that Xinjiang produces Radix Glycyrrhizae, which is often mingled in the seed in other places of production, to be sold.And from Glycyrrhiza Seeds
It is difficult to accurately distinguish Xinjiang production Radix Glycyrrhizae in terms of mode of appearance.
Molecular marking technique such as RAPD (Random Amplified Polymorphic DNA, randomly amplified polymorphic DNA
Label), AFLP (Amplified Fragment Length Polymorphism, amplified fragment length polymorphism), SSR
Reproducibility of results such as (Simple Sequence Repeat, simple sequence repeat markers) are poor, heavy workload, at high cost.
Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to generation in chromosome base
Because in group level single nucleotide acid variation caused by DNA sequence polymorphism.In very long evolutionary process, organism is formd certainly
The distinctive DNA base sequence of body, compares between different plant species or the interracial SNP difference of same species different places, it will be appreciated that
The bioinformatics of affiliation and evolution between species, so that the kind to different plant species or same species different places carries out
Fine identification and classification.The difference of single nucleotide acid is detected from molecular level, Single nuclear polymorphism label can
Help distinguish between the difference of inhereditary material between individual.
Summary of the invention
The present invention relates to a kind of, and identifying or assisting in based on single nucleotide polymorphism identifies the method that Xinjiang produces Radix Glycyrrhizae,
With the molecular marking technique of molecular biology, DNA extraction, PCR amplification, primer are carried out to the Radix Glycyrrhizae plant sample of different sources
Screening and bioinformatic analysis find and quick and precisely identify the method that Xinjiang produces Radix Glycyrrhizae.
Inventor is by largely reading document and the modes such as investigation determine the main producing region of Radix Glycyrrhizae on the spot.Radix Glycyrrhizae is main at present
The ground such as Xinjiang, Gansu, Ningxia, Shaanxi, the Inner Mongol are distributed in, it is wild to have resource distribution with cultivation.By going deep into Radix Glycyrrhizae main product
Area, with local large plantation family link up, acquire a large amount of certified products Radix Glycyrrhizae it is wild with cultivation sample.
By carrying out DNA extraction to the sample of acquisition, PCR amplification is carried out with different aligning primers, and be sequenced, passed through
Bioinformatic analysis aligned sequences, the sequence of screening include karyogene sequence ITS (Internal transcribed
Spacer, the Internal Transcribed Spacer) and ITS2 (Internal transcribed spacer 2), chloroplast gene sequence psbA-
TrnH (being primarily referred to as psbA and tRNA-His intergenic spacer region spacer sequence), rbcL (ribulose
1,5-bisphosphate carboxylase/oxygenase large subunit)、matK(maturase K)。
Sequence alignment and analysis are carried out with MEGA software, the accurate primer sequence for identifying Xinjiang and producing Radix Glycyrrhizae can be stablized by finding
(in a certain site of the sequence or certain several site, Xinjiang produces Radix Glycyrrhizae to column and the base of other place of production samples is different, and respectively
Unanimously).It is compared and is screened by final data, Xinjiang can be produced Radix Glycyrrhizae and its by discovery Chloroplast gene psbA-trnH sequence
Its place of production Radix Glycyrrhizae is identified.By sequence alignment analysis, the 270 site (psbA-trnH in psbA-trnH sequence are found
Sequence is independent sequence, have its fix component part, be psbA, psbA-trnH intergenic region, trnH,
Although the length of each component part is different between different plant species, the length of the psbA-trnH sequence of same species or its sibling species
Degree is almost the same.And after passing through MEGA software progress sequence alignment, the base of every sequence is corresponding, guarantees every
Sequence is all the generation base mutation at 270 sites), mutating alkali yl (the Single Nucleotide of regularity is presented
Polymorphisms, single nucleotide polymorphism, SNP variant sites).Xinjiang produces Radix Glycyrrhizae in 270 sites of psbA-trnH sequence
For T base, the Radix Glycyrrhizae in other places of production is C base (as shown in Figure 1) in 270 sites of psbA-trnH sequence.
Based on this, the present invention provide it is a kind of based on single nucleotide polymorphism identifying or assisting in identify Xinjiang produce Radix Glycyrrhizae
Method comprising following steps:
1) DNA of Radix Glycyrrhizae to be measured is extracted；
2) by sequencing, if the 270th site chloroplast gene sequence psbA-trnH is T base, which is new
Boundary produces Radix Glycyrrhizae, if the 270th site chloroplast gene sequence psbA-trnH is C base, which is that other places of production are sweet
Grass.
In the preferred embodiment of the present invention, primer pair can be added and carry out PCR amplification, obtain amplified production, then
Amplified production is sequenced.Preferably, using the fwdPA/revTH primer of psbA-trnH sequence, (fwdPA/revTH draws
Object to) to template carry out PCR amplification.
In the preferred embodiment of the present invention, primer pair can be added and carry out PCR amplification, obtain amplified production, then
Electrophoresis detection is carried out to amplified production.Preferably, amplified production is the DNA fragmentation of about 200bp.
The present invention relates to for identifying or assisting in the primer pair for identifying Xinjiang and producing Radix Glycyrrhizae, can be selected from: primer pair 1 is drawn
Object is to 2 and primer pair 3, wherein the sequence of primer pair 1 is as shown in SEQ ID NO:1 and SEQ ID NO:2, the sequence of primer pair 2
As shown in SEQ ID NO:3 and SEQ ID NO:4, the sequence of primer pair 3 is as shown in SEQ ID NO:5 and SEQ ID NO:6.
The present invention relates to a kind of for identifying or assisting in the method for identifying Xinjiang and producing Radix Glycyrrhizae comprising following steps:
1) DNA of Radix Glycyrrhizae to be measured is extracted as template；
2) PCR amplification is carried out as PCR amplification primer using at least one primer pair, obtains amplified production；
3) amplified production is detected.
Further, the detection for being detected as molecular level.It can be such as hybridization, genetic chip, PCR detection.PCR
Detect it is simple and easy to do, it is therefore preferable that for PCR detect.
In the preferred embodiment of the present invention, the primer pair is selected from: primer pair 1, primer pair 2 and primer pair 3,
In, the sequence of primer pair 1 as shown in SEQ ID NO:1 and SEQ ID NO:2, the sequence of primer pair 2 such as SEQ ID NO:3 and
Shown in SEQ ID NO:4, the sequence of primer pair 3 is as shown in SEQ ID NO:5 and SEQ ID NO:6.
In the preferred embodiment of the present invention, the PCR condition of primer pair 1 are as follows: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation
30s, 50 DEG C of annealing 1min, 72 DEG C of extension 1min, 35 recycle；72 DEG C of extension 10min.
In the preferred embodiment of the present invention, the PCR condition of primer pair 2 are as follows: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation
30s, 50 DEG C of annealing 1min, 72 DEG C of extension 1min, 35 recycle；72 DEG C of extension 10min.
In the preferred embodiment of the present invention, the PCR condition of primer pair 3 are as follows: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation
30s, 48 DEG C of annealing 1min, 72 DEG C of extension 1min, 35 recycle；72 DEG C of extension 10min.
In the preferred embodiment of the present invention, electrophoresis can be agglomerated to detect the amplified production by agarose, if
There is amplified band in electrophoresis showed, then shows that the Radix Glycyrrhizae to be measured is that Xinjiang produces Radix Glycyrrhizae.Preferably, point of the amplified band
Son amount is about 200bp.
In the preferred embodiment of the present invention, fluorescent dye can be added in the reaction system after amplification, such as glimmering
Photoinitiator dye SYBR Green I, if detecting, the system containing fluorescent dye generates green fluorescence, shows that this is to be measured sweet
Grass is that Xinjiang produces Radix Glycyrrhizae.Preferably, it is under 365nm ultraviolet wavelength that whether the system containing fluorescent dye, which generates green fluorescence,
It is detected.
The present invention also provides the PCR reagent that Xinjiang produces Radix Glycyrrhizae or auxiliary identifies Xinjiang production Radix Glycyrrhizae is identified, can contain
At least one above-mentioned primer pair, the PCR reagent can be used for detecting whether Radix Glycyrrhizae to be measured is that Xinjiang produces Radix Glycyrrhizae or whether contains
Xinjiang produces Radix Glycyrrhizae.
It, can be containing extremely the present invention also provides the kit that Xinjiang produces Radix Glycyrrhizae or auxiliary identifies Xinjiang production Radix Glycyrrhizae is identified
Few a kind of above-mentioned primer pair or at least one above-mentioned PCR reagent.The kit produces Radix Glycyrrhizae for identification Xinjiang and provides just
Benefit.
It include PCR reaction buffer, archaeal dna polymerase and dNTP in the kit in the preferred embodiment of the present invention.
In the preferred embodiment of the present invention, fluorescent dye, such as fluorescent dye can also be contained in the kit
SYBR Green I。
Detailed description of the invention
The software screenshot of Fig. 1 different sources Radix Glycyrrhizae psbA-trnH sequence SNP variant sites
Fig. 2 primer pair 1 identifies electrophoresis detection figure when different sources Radix Glycyrrhizae
Fig. 3 primer pair 2 identifies electrophoresis detection figure when different sources Radix Glycyrrhizae
Fig. 4 primer pair 3 identifies electrophoresis detection figure when different sources Radix Glycyrrhizae
Described herein and claimed invention content is not limited to the model of embodiment in detail below disclosed herein
It encloses, these embodiments are intended only to illustrate several aspects of the invention.
The screening of 1 single nucleotide polymorphism of embodiment
The Radix Glycyrrhizae for acquiring standard from Xinjiang, Gansu, the Inner Mongol, the Ningxia main producing region Deng Di is wild to amount to 302 with cultivation sample
Part, it can typically represent the sample type of current each main producing region.
The details of the Radix Glycyrrhizae standard items of acquisition see the table below 1:
Collecting sample is acquisition (attached GPS information) on the spot
1.1 Genome DNA extraction
Each place of production Radix Glycyrrhizae sample of acquisition is dried using silica gel, after being completely dried, uses small steel ball and ball milling
Instrument (German FRITSCH, PULVERISETTE 6) smashes and grinds sample.Using RNA isolation kit (CTAB method, Tiangeng are biochemical,
DP305 kit) extract plant sample genome DNA.Specific step is as follows:
1) ground sample powder is transferred quickly in the EP pipe of 2.0mL, the buffer GP1 of 65 DEG C of preheatings is added
700 μ l, 1 μ l of mercaptoethanol；The pipe lid top EP is sealed with Parafilm sealed membrane, after mixing of turning upside down, is put into
65 DEG C of water-bath 30min, during water-bath, constantly reverse EP pipe is for several times.
2) 700 μ l chloroforms are added: isoamyl alcohol (24:1) mixed liquor mixes well, and 12000rpm is centrifuged 5min.
3) supernatant separated after centrifugation is carefully transferred in new 2.0EP pipe, 700 μ l isopropanols is added and (put -20 DEG C
Refrigerator saves, and cryogenic effect is more preferably), it mixes well.
4) liquid after mixing is transferred in two times in adsorption column CB3,12000rpm is centrifuged 30s, discards in collecting pipe
Waste liquid.
5) 500 μ l buffer GD (using the preceding determining dehydrated alcohol that prescribed volume is added) is added into adsorption column CB3,
12000rpm is centrifuged 30s, discards the waste liquid in collecting pipe.
6) 700 μ l rinsing liquid PW are added into adsorption column CB3, (using the preceding determining dehydrated alcohol that prescribed volume is added),
12000rpm is centrifuged 30s, discards the waste liquid in collecting pipe.
7) 6 steps are repeated.
8) adsorption column CB3 is put back in collecting pipe, 12000rpm is centrifuged 2min, outwells the waste liquid in collecting pipe.It will absorption
Column CB3 is put into new 1.5mL EP pipe, is set and is placed at room temperature for several minutes, until thoroughly drying the remaining rinsing in adsorption column CB3
Liquid (using no ethanol flavor as judgment criteria).
9) to 100 μ l eluent TE are vacantly added until filter membrane among adsorption column CB3,3-5 minutes are placed at room temperature for, so that washing
The de- abundant eluted dna of liquid.
10) the 1.5mLEP pipe 12000rpm for being placed with adsorption column CB3 is centrifuged 2min, solution is collected into 1.5mLEP pipe
Interior, -20 DEG C save backup.
1.2 primer screenings and PCR amplification
Primer is selected, PCR amplification and electrophoresis detection, pcr amplification reaction body are carried out to the total DNA of different sources Radix Glycyrrhizae sample
System is 25 μ l, and group becomes ddH28.5 μ l, 2 × Taq PCR MIX of O 12.5 μ l, each 1 μ l of upstream and downstream primer, 2 μ l of DNA profiling.
Each primer sequence and PCR reaction condition are respectively as follows:
ITS2 sequence, primer (S2F:ATGCGATACTTGGTGTGAAT, S3R:GACGCTTCTCCAGACTACAAT),
The primer pair sequence respectively as shown in SEQ ID NO:7 and SEQ ID NO:8,
94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 30 seconds, and 56 DEG C are annealed 30 seconds, and 72 DEG C extend 45 seconds, 40 circulations；72℃
Extend 10 minutes.
ITS sequence, primer (5F:GGAAGTAAAAGTCGTAACAAGG, 4R:TCCTCCGCTTATTGATATGC) should
Primer pair sequence respectively as shown in SEQ ID NO:9 and SEQ ID NO:10,
94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 1 minute, and 50 DEG C are annealed 1 minute, 72 DEG C of extensions, 1.5 minutes (each circulations
Increase by 3 seconds), 30 circulations；72 DEG C extend 7 minutes.
PsbA-trnH sequence, primer (fwdPA:GTTATGCATGAACGTAATGCTC, revTH:
CGCGCATGGTGGATTCACAATC C), the primer pair sequence respectively as shown in SEQ ID NO:11 and SEQ ID NO:12,
94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 1 minute, and 55 DEG C are annealed 1 minute, and 72 DEG C extend 1.5 minutes, 30 circulations；
72 DEG C extend 7 minutes.
MatK sequence, primer (KIM_3F:CGTACAGTACTTTTGTGTTTACGAG, KIM_1R:
ACCCAGTCCATCTGGAA ATCTTGGTTC), the primer pair sequence is respectively such as SEQ ID NO:13 and SEQ ID NO:14 institute
Show,
94 DEG C initial denaturation 1 minute；94 DEG C are denaturalized 30 seconds, and 52 DEG C are annealed 20 seconds, and 72 DEG C extend 50 seconds, 35 circulations；72℃
Extend 5 minutes.
RbcL sequence, primer (1f:ATGTCACCACAAACAGAAAC, 724r:TCGCATGTACCTGCAGTAGC) should
Primer pair sequence respectively as shown in SEQ ID NO:15 and SEQ ID NO:16,
95 DEG C initial denaturation 2 minutes；94 DEG C are denaturalized 1 minute, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute, 34 circulations；72
DEG C extend 7 minutes.
PCR reaction product electrophoresis detection: 1% Ago-Gel of configuration (0.6g agarose 0.5 × tbe buffer liquid of+60mL,
Microwave accelerates dissolution；1 μ l nucleic acid dye GeneGreen is added, is poured into when being cooled to 65 DEG C or so in the glue box for placing comb；
10-20 minutes are placed at room temperature for, gel extracts comb after thoroughly solidifying, and gel is put into electrophoresis tank, the tbe buffer in electrophoresis tank
Liquid need to not cross gel；), PCR product is clicked and entered in gel, while clicking and entering DNA Marker (DL2000), adjusts electrophoresis apparatus voltage
To 100-140V, the time is 30 minutes, starts electrophoresis.After electrophoresis, gel is put into gel imager, is observed under ultraviolet lamp
Electrophoretic band, such as every part of sample have single and bright band, illustrate PCR amplification success, can carry out subsequent examining order.
1.3 sequencings and sequence comparing analysis
Whole PCR products are clicked and entered and carry out electrophoresis in Ago-Gel, and the pillar location in gel is carried out to cut glue time
It receives, and upper machine sequencing is carried out to product after the recovery.
After the sequencing is completed, sequence assembly is carried out with CodonCode Aligener software to the sequence of return, removed low
Quality region sequence；Sequence alignment and analysis are carried out with MEGA software, the accurate Xinjiang that identifies can be stablized by, which finding, produces drawing for Radix Glycyrrhizae
Object sequence.By sequence alignment analysis, it is found that the mutating alkali yl of regularity is presented in 270 sites in psbA-trnH sequence
(Single Nucleotide Polymorphisms, single nucleotide polymorphism, SNP variant sites).The Radix Glycyrrhizae in other places of production exists
270 sites of psbA-trnH sequence are C base, and as shown in Figure 1, producing Radix Glycyrrhizae at 270 of psbA-trnH sequence in Xinjiang
Point is T base.
Embodiment 2 is based on single nucleotide polymorphism and identifies to Xinjiang production Radix Glycyrrhizae
2.1 Genome DNA extraction
The dry sample of Radix Glycyrrhizae to be measured is taken, about 30mg can be directly weighed after removing surface contaminant, be put into the 1.5mL of sterilizing
In centrifuge tube, small steel ball after two sterilizings are added is put into ball milling instrument 1500rpm and grinds 3-5 minutes；Fresh sample can weigh
About 0.5g removes surface contaminant, is added in clean mortar, is fully ground into powder with liquid nitrogen, take it is appropriate be added 1.5mL from
In heart pipe；
250 μ l nucleus lysates are added (to be mainly used for the cell wall cracking in sample releasing intracellular heredity
Substance), mixing fullys shake in 5 μ l RNA enzyme solution；
20 μ l Proteinase Ks are added, mixing fullys shake；
It places it in 65 DEG C of water-baths water-bath 1 hour (period reverses mixing for several times repeatedly)；
250 μ l lysis buffers are added, are fully transferred to after mixing well in purifying adsorption column, 12000rpm is centrifuged 5 points
Clock；
Remove the waste liquid in collecting pipe, is added 700 μ l rinsing liquids (dehydrated alcohol has been added using preceding determination), 12000rpm
Centrifugation 40 seconds；
Remove the waste liquid in collecting pipe, repeats step 6) 2 times；
Remove the waste liquid in collecting pipe, blank pipe 12000rpm is centrifuged 2 minutes, will purifying adsorption column be put into new 1.5mL from
In heart pipe, 5 minutes are placed at room temperature for, can be distributed entirely to ethyl alcohol；
The elution buffer of 100 μ l sterilizing is vacantly added on the center filter membrane of purifying adsorption column, is placed at room temperature for 2 minutes,
12000rpm is centrifuged 2 minutes up to sample DNA template, puts -20 DEG C and saves backup.
2.2 PCR amplification
25 μ l of PCR reaction system is configured, is put into 0.2mLPCR pipe, system includes 12.5 μ l 2 × Taq PCR Mix,
2 μ l of DNA profiling, primer (up/down trip, 2.5 μM) each 1 μ l, sterilize 8.5 μ l of distilled water, to different sources Radix Glycyrrhizae sample to be measured
Total DNA carries out PCR amplification and electrophoresis detection.
Primer sequence and PCR reaction condition are respectively as follows:
PsbA-trnH sequence, primer (fwdPA:GTTATGCATGAACGTAATGCTC, revTH:
CGCGCATGGTGGATTCACAATCC),
94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 1 minute, and 55 DEG C are annealed 1 minute, and 72 DEG C extend 1.5 minutes, 30 circulations；
72 DEG C extend 7 minutes.
2.3 sequencing analysis
PCR product is clicked and entered and carries out electrophoresis in Ago-Gel, and gel extraction is carried out to the pillar location in gel, and
Upper machine sequencing is carried out to product after the recovery.
If the 270th site of psbA-trnH sequence is T base, which is that Xinjiang produces Radix Glycyrrhizae；If psbA-trnH
The 270th site of sequence is C base, then the Radix Glycyrrhizae to be measured is the Radix Glycyrrhizae in other places of production.
3 specific primer of embodiment
According to the stabilization SNP variant sites for identifying Xinjiang and producing Radix Glycyrrhizae can be stablized, the spy for identifying Xinjiang and producing Radix Glycyrrhizae is designed with this
Specific primer.Select the complementary series of 20bp length as upstream primer sequence at the upstream 30-49bp of psbA-trnH sequence
Column, by taking Xinjiang produces Radix Glycyrrhizae as an example, 270 site of psbA-trnH sequence is T base, before 270 sites of psbA-trnH sequence
After separately design three groups of primers as downstream primer, respectively 250-270bp, 260-280bp, 270-291bp.This three Duan Xulie
Reverse complementary sequence respectively correspond three groups of the downstream primer sequence of specific primer.This process can pass through software primer 6.0
Realize conversion or the manual modification of sequence and primer sequence.
Three pairs of primer pairs are respectively as follows:
The sequence of primer pair 1 such as SEQ ID NO:1 (5 '-CTTCGAGGTAGATATTTACC-3 ') and SEQ ID NO:2
Shown in (5 '-GCGCGTCTTCTAAAATACGAG-3 ')；
The sequence of primer pair 2 such as SEQ ID NO:3 (5 '-CTTCGAGGTAGATATTTACC-3 ') and SEQ ID NO:4
Shown in (5 '-TTAAGTTCTTGCGCGTCTTCT-3 ')；
The sequence of primer pair 3 such as SEQ ID NO:5 (5 '-CTTCGAGGTAGATATTTACC-3 ') and SEQ ID NO:6
Shown in (5 '-TTTCTTCTCTTTTAAGTTCTTG-3 ').
Primer sequence is analyzed with 6.0 software of primer, determine annealing temperature and carries out assay optimization, finally
Determine the PCR response procedures of each primer pair are as follows:
The PCR condition of primer pair 1 are as follows: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, 50 DEG C of annealing 1min, 72 DEG C extend
1min, 35 circulations；72 DEG C of extension 10min；
The PCR condition of primer pair 2 are as follows: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, 50 DEG C of annealing 1min, 72 DEG C extend
1min, 35 circulations；72 DEG C of extension 10min；
The PCR condition of primer pair 3 are as follows: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, 48 DEG C of annealing 1min, 72 DEG C extend
1min, 35 circulations；72 DEG C of extension 10min.
Embodiment 4 is by specific primer to the method for identifying Xinjiang production Radix Glycyrrhizae
It mainly includes that DNA extracts part, the amplification part containing PCR reagent that specificity, which identifies Xinjiang to produce the identification system of Radix Glycyrrhizae,
Three parts are detected with agarose gel electrophoresis, wherein the PCR reagent contains specific primer.It mainly includes cell that DNA, which extracts part,
Karyorhexis liquid, lysate buffer, RNA enzyme solution, Proteinase K, rinsing liquid (dehydrated alcohol need to be added when use) sterile are washed
De- buffer, purifying adsorption column, collecting pipe, 1.5mL sterile centrifugation tube etc..PCR amplification part mainly includes 2 × Taq PCR
Mix, specific primer (up/down trip), standard Xinjiang produce Radix Glycyrrhizae DNA (positive control), the other place of production Radix Glycyrrhizae DNA of standard (feminine gender
Control), sterilizing distilled water etc..Agarose gel electrophoresis detection part includes agarose, 50 × tbe buffer liquid, DNA marker
(DNA molecular amount label) and nucleic acid dye.
Detection method:
4.1 Genome DNA extraction
The dry sample of Radix Glycyrrhizae to be measured is taken, about 30mg can be directly weighed after removing surface contaminant, be put into sterilizing
In 1.5mL centrifuge tube, small steel ball after two sterilizings are added is put into ball milling instrument 1500rpm and grinds 3-5 minutes；Fresh sample
About 0.5g can be weighed, surface contaminant is removed, is added in clean mortar, is fully ground into powder with liquid nitrogen, take appropriate addition
In 1.5mL centrifuge tube；
250 μ l nucleus lysates are added (to be mainly used for the cell wall cracking in sample releasing intracellular heredity
Substance), mixing fullys shake in 5 μ l RNA enzyme solution；
20 μ l Proteinase Ks are added, mixing fullys shake；
It places it in 65 DEG C of water-baths water-bath 1 hour (period reverses mixing for several times repeatedly)；
250 μ l lysis buffers are added, are fully transferred to after mixing well in purifying adsorption column, 12000rpm is centrifuged 5 points
Clock；
Remove the waste liquid in collecting pipe, is added 700 μ l rinsing liquids (dehydrated alcohol has been added using preceding determination), 12000rpm
Centrifugation 40 seconds；
Remove the waste liquid in collecting pipe, repeats step 6) 2 times；
Remove the waste liquid in collecting pipe, blank pipe 12000rpm is centrifuged 2 minutes, will purifying adsorption column be put into new 1.5mL from
In heart pipe, 5 minutes are placed at room temperature for, can be distributed entirely to ethyl alcohol；
The elution buffer of 100 μ l sterilizing is vacantly added on the center filter membrane of purifying adsorption column, is placed at room temperature for 2 minutes,
12000rpm is centrifuged 2 minutes up to sample DNA template, puts -20 DEG C and saves backup.
4.2 PCR amplification
25 μ l of PCR reaction system is configured, is put into 0.2mLPCR pipe, system includes 12.5 μ l 2 × Taq PCR Mix,
2 μ l of DNA profiling, specific primer (up/down trip, 2.5 μM) each 1 μ l, sterilize 8.5 μ l of distilled water.Other than sample DNA template,
Positive control, negative control and blank control sample PCR reaction system, method is separately configured to be same as above, in addition to DNA profiling is different,
Blank control replaces DNA profiling with sterilizing distilled water.
System solution is mixed well, after being centrifuged the several seconds with micro centrifuge, is put into PCR instrument.
PCR response procedures are corresponding with specific primer, are respectively as follows:
The PCR condition of primer pair 1 are as follows: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, 50 DEG C of annealing 1min, 72 DEG C extend
1min, 35 circulations；72 DEG C of extension 10min；
The PCR condition of primer pair 2 are as follows: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, 50 DEG C of annealing 1min, 72 DEG C extend
1min, 35 circulations；72 DEG C of extension 10min；
The PCR condition of primer pair 3 are as follows: 95 DEG C of initial denaturation 4min；94 DEG C of denaturation 30s, 48 DEG C of annealing 1min, 72 DEG C extend
1min, 35 circulations；72 DEG C of extension 10min.
After reaction to PCR, it is put into -4 DEG C after PCR product being taken out to save backup, or carries out Ago-Gel immediately
Electrophoresis detection
4.3 electrophoresis detection
1) Ago-Gel makes
0.6g agarose is weighed, is added in 0.5 × tbe buffer liquid of 60mL and (is 50 × tbe buffer liquid in kit, is suitble to
Long-term preservation is directly diluted to 0.5 × tbe buffer liquid using Shi Keyong distilled water and uses, it is proposed that is ready-to-use), microwave is added
Heating accelerates solution (high fire 1-2 minutes) in furnace, and after agarose is completely dissolved and when temperature is down to 65 DEG C or so, 1 μ l is added
Nucleic acid dye is poured into after slowly shaking up and is inserted in the glue box of comb, solidifies at room temperature.
2) electrophoresis detection
0.5 fresh × tbe buffer liquid is added in the electrophoresis tank of electrophoresis apparatus, the gel solidified is put into electrophoresis tank
(buffer need to not cross gel), the well side of gel is placed on the cathode of electrophoresis tank.5 μ l are sequentially added from one end of well
DNA marker, positive control, negative control, blank control, detection sample DNA PCR product, by 5V/cm constant pressure carry out
Electrophoresis 30 minutes.
3) gel imaging system is analyzed
After electrophoresis, Ago-Gel is put into gel imager observe and is taken pictures.It is constantly exposed using ultraviolet lamp
For light until band on glue figure is clear, preservation running gel figure result is electronic document, and observes and records electrophoresis detection result.
4) Analysis of test results
If negative control and blank control do not occur band, there is the amplified band of 200bp or so in positive control, such as
There is the amplified band of corresponding 200bp or so in sample to be tested, can determine that sample to be tested is positive findings (Xinjiang produces Radix Glycyrrhizae)；Such as
Sample to be tested does not occur the amplified band of 200bp corresponding with positive control or so, can determine that the sample to be tested is not Xinjiang
Produce Radix Glycyrrhizae.
PCR amplification is carried out with master sample of three pairs of specific primers to Xinjiang and other place of production Radix Glycyrrhizaes and agarose is solidifying
Gel electrophoresis detection, three pairs of specific primers of the present invention can specific amplification Xinjiang produce Radix Glycyrrhizae sample, and cannot expand
Other place of production Radix Glycyrrhizae samples.Electrophoresis detection map such as Fig. 2, Fig. 3 that the specific Radix Glycyrrhizae sample of three pairs of primer pairs is expanded and
Shown in Fig. 4, there is the amplification of corresponding about 200bp in the electrophoresis detection for the standard items that the Xinjiang in embodiment 1 produces Radix Glycyrrhizae
Band, and the electrophoresis detection of the Radix Glycyrrhizae standard items in other places of production in embodiment 1 does not occur the expansion of corresponding about 200bp
Increase band.Whether three pairs of primer pairs of the present invention can specific detection Radix Glycyrrhizae to be measured be quickly that is produced from Xinjiang as a result,
Radix Glycyrrhizae.
SEQUENCE LISTING
<110>Chinese medicine Co., Ltd
<120>primer pair and its application for identifying Radix Glycyrrhizae are identified or assisting in
<130>
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
cttcgaggta gatatttacc 20
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<400> 2
gcgcgtcttc taaaatacga g 21
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
cttcgaggta gatatttacc 20
<210> 4
<211> 21
<212> DNA
<213>artificial sequence
<400> 4
ttaagttctt gcgcgtcttc t 21
<210> 5
<211> 20
<212> DNA
<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 (10)
1. it is a kind of for identifying or assisting in the primer pair for identifying Radix Glycyrrhizae, it is selected from primer pair 1, primer pair 2 and primer pair 3, wherein
The sequence of primer pair 1 is as shown in SEQ ID NO:1 and SEQ ID NO:2, the sequence of primer pair 2 such as SEQ ID NO:3 and SEQ
Shown in ID NO:4, the sequence of primer pair 3 is as shown in SEQ ID NO:5 and SEQ ID NO:6.
2. primer pair described in claim 1 is used to identify or assisting in identification Xinjiang and produces Radix Glycyrrhizae.
3. a kind of for identifying the method for Radix Glycyrrhizae comprising following steps:
1) DNA of Radix Glycyrrhizae to be measured is extracted as template；
2) it uses at least one selected from primer pair described in claim 1 for PCR amplification primer, carries out PCR amplification, expanded
Product；
3) amplified production is detected.
4. method as claimed in claim 3, wherein agglomerating electrophoresis by agarose to detect the amplified production, if electrophoresis showed
There is amplified band, then shows that the Radix Glycyrrhizae to be measured is that Xinjiang produces Radix Glycyrrhizae.
5. method as claimed in claim 4, wherein the molecular weight of the amplified band is about 200bp.
6. method as claimed in claim 3, wherein fluorescent dye SYBR Green I is added in reaction system after amplification, if
It detects that the system containing fluorescent dye generates green fluorescence, then shows that the Radix Glycyrrhizae to be measured is that Xinjiang produces Radix Glycyrrhizae.
7. a kind of identify or assisting in the PCR reagent for identifying Radix Glycyrrhizae, it includes at least one primer pairs described in claim 1.
8. a kind of for identifying or assisting in the kit for identifying Radix Glycyrrhizae, it includes at least one primer pairs described in claim 1
Or at least one PCR reagent as claimed in claim 7.
9. kit according to any one of claims 8 comprising PCR reaction buffer, archaeal dna polymerase and dNTP.
10. reagent described in primer pair claimed in claims 1-2, PCR reagent as claimed in claim 7 or claim 8-9
Box is identifying or assisting in the application identified in Xinjiang production Radix Glycyrrhizae.
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| CN201810747468.5A CN109735644B (en) | 2018-07-09 | 2018-07-09 | Primer pair for identifying or assisting in identifying liquorice and application thereof |
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Cited By (1)
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| CN112322781A (en) * | 2021-01-05 | 2021-02-05 | 中国中药有限公司 | SNP molecular marker for identifying liquorice produced in Gansu province and method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112322781A (en) * | 2021-01-05 | 2021-02-05 | 中国中药有限公司 | SNP molecular marker for identifying liquorice produced in Gansu province and method and application thereof |
| 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 |
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| CN109735644B (en) | 2022-04-08 |
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